Nicotinamide mononucleotide derivatives and their uses

ABSTRACT

The invention relates to compositions of nicotinamide mononucleotide derivatives and their methods of use. The invention also relates to methods of preparing nicotinamide mononucleotide derivatives. The invention relates to pharmaceutical compositions and nutritional supplements containing a nicotinamide mononucleotide derivative. The invention relates to methods of using nicotinamide mononucleotide derivatives that promote the increase of intracellular levels of nicotinamide adenine dinucleotide (NAD+) in cells and tissues for treating diseases and improving cell and tissue survival.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/512,388, which is the U.S. national phase of International PatentApplication No. PCT/US2016/045855, filed Aug. 5, 2016, which claims thebenefit of priority of U.S. Patent Application Ser. No. 62/201,447,filed Aug. 5, 2015, the contents of which are hereby incorporated byreference in their entirety.

BACKGROUND OF THE INVENTION

Nicotinamide adenine dinucleotide (NAD) and its derivative compounds areknown as essential coenzymes in cellular redox reactions in all livingorganisms. Several lines of evidence have also shown that NADparticipates in a number of important signaling pathways in mammaliancells, including poly(ADP-ribosyl)ation in DNA repair (Menissier deMurcia et al., EMBO J., (2003) 22, 2255-2263), mono-ADP-ribosylation inthe immune response and G protein-coupled signaling (Corda and DiGirolamo, EMBO J., (2003) 22, 1953-8), and the synthesis of cyclicADP-ribose and nicotinate adenine dinucleotide phosphate (NAADP) inintracellular calcium signaling (Lee, Annu. Rev. Pharmacol. Toxicol.,(2001) 41, 317-345). Recently, it has also been shown that NAD and itsderivatives play an important role in transcriptional regulation (Linand Guarente, Curr. Opin. Cell. Biol., (2003) 15, 241-246). Inparticular, the discovery of Sir2 NAD-dependent deacetylase activity(e.g., Imai et al., Nature, (2000) 403, 795-800; Landry et al., Biochem.Biophys. Res. Commun., (2000) 278, 685-690; Smith et al., Proc. Natl.Acad. Sci. USA, (2000) 97, 6658-6663) drew attention to this new role ofNAD.

The Sir2 family of proteins consumes NAD for its deacetylase activityand regulates transcription by deacetylating histones and a number ofother transcription regulators.

Because of this absolute requirement for NAD, it has been proposed thatSir2 proteins function as energy sensors that convert the energy statusof cells to the transcriptional regulatory status of genes (Imai et al.,Nature, (2000) 403, 795-800; Imai et al., Cold Spring Harbor Symp.Quant. Biol., (2000) 65, 297-302). Sir2 proteins produce nicotinamideand O-acetyl-ADP-ribose in addition to the deacetylated proteinsubstrates in their deacetylation reaction (Moazed, Curr. Opin. Cell.Biol., (2001) 13, 232-238; Denu, Trends Biochem. Sci., (2003) 28, 41-48;see also FIG. 1), and nicotinamide is eventually recycled into NADbiosynthesis. Unlike other NAD-dependent biochemical reactions, theNAD-dependent deacetylase activity of the Sir2 family of proteins isgenerally highly conserved from bacteria to mammals (Frye, Biochem.Biophys. Res. Commun., (2000) 273, 793-798), suggesting that theconnection between NAD and Sir2 proteins is ancient and fundamental. Inmammals, the Sir2 ortholog, Sirt1/Sir2α, has been shown to regulatemetabolism in response to nutrient availability (Bordone and Guarente,Nat. Rev. Mol. Cell Biol., (2005) 6, 298-305). In adipocytes, Sirt1triggers lipolysis and promotes free fatty acid mobilization byrepressing PPAR-gamma, a nuclear receptor that promotes adipogenesis(Picard et al., Nature, (2004) 429, 771-776). In hepatocytes, Sirt1regulates the gluconeogenic and glycolytic pathways in response tofasting by interacting with and deacetylating PGC-la, a keytranscriptional regulator of glucose production in the liver (Rodgers etal., Nature, (2005) 434, 113-118). Additionally, Sirt1 promotes insulinsecretion in pancreatic beta cells in response to high glucose partly byrepressing Ucp2 expression and increasing cellular ATP levels (Moynihanet al., Cell Metab., (2005) 2, 105-117). While little is known about theregulation of NAD biosynthesis in mammals, NAD biosynthesis may play arole in the regulation of metabolic responses by altering the activityof certain NAD-dependent enzymes such as Sirt1 in a variety of organsand/or tissues.

The NAD biosynthesis pathways have been characterized in prokaryotes byusing Escherichia coli and Salmonella typhimurium (Penfound and Foster,Biosynthesis and recycling of NAD, in Escherichia coli and Salmonella:Cellular and Molecular Biology, p. 721-730, ed. Neidhardt, F. C., 1996,ASM Press: Washington, D.C.) and recently in yeast (Lin and Guarente,Curr. Opin. Cell. Biol., (2003) 15, 241-246; Denu, Trends Biochem. Sci.,(2003) 28, 41-48). In prokaryotes and lower eukaryotes, NAD issynthesized by the de novo pathway via quinolinic acid and by thesalvage pathway via nicotinic acid (Penfound and Foster, id.) In yeast,the de novo pathway begins with tryptophan, which is converted tonicotinic acid mononucleotide (NaMN) through six enzymatic steps and onenon-enzymatic reaction (Lin and Guarente, Curr. Opin. Cell. Biol.,(2003) 15, 241-246). Two genes, BNA1 and QPT1, have been characterizedin this pathway in yeast. At the step of NaMN synthesis, the de novopathway converges with the salvage pathway. The salvage pathway beginswith the breakdown of NAD into nicotinamide and O-acetyl-ADP-ribose,which is mainly catalyzed by the Sir2 proteins in yeast. Nicotinamide isthen deamidated to nicotinic acid by a nicotinamidase encoded by thePNC1 gene. Nicotinic acid phosphoribosyltransferase (Npt), encoded bythe NPT1 gene, converts nicotinic acid to NaMN, which is eventuallyconverted to NAD through the sequential reactions ofnicotinamide/nicotinic acid mononucleotide adenylyltransferase (encodedby NMA1 and/or NMA2) and NAD synthetase (encoded by QNS1).

Many aspects of mammalian behavior and physiology are coordinatedthrough interconnected networks of 24-hour central and peripheraloscillators that synchronize cycles of fuel storage and utilization tomaintain organismal homeostasis. In mice, circadian disruption has beentied to metabolic disturbance (F. W. Turek et al., Science 308, 1043(2005); R. D. Rudic et al., PLoS Biol. 2, e377 (2004)), whileconversely, high-fat diet alters both behavioral and molecular rhythms(A. Kohsaka et al., Cell Metab. 6, 414 (2007); M. Barnea, Z. Madar, O.Froy, Endocrinology 150, 161 (2009)). The underlying mechanism of themammalian clock consists of a transcription-translation feedback loop inwhich CLOCK and BMAL1 activate transcription of Cryptochrome (Cry1 and2) and Period (Per1, 2, and 3), leading to subsequent repression ofCLOCK:BMAL1 by CRY and PER proteins (J. S. Takahashi, H. K. Hong, C. H.Ko, E. L. McDearmon, Nat. Rev. Genet. 9, 764 (2008)). An additionalfeedback loop involves the transcriptional regulation of Bmal1 by ROR□and REV-ERB□ (N. Preitner et al., Cell 110, 251 (2002); T. K. Sato etal., Neuron 43, 527 (2004)). Previous studies have also implicated arole for cellular NAD+ in the regulation of CLOCK and NPAS2 activity (J.Rutter, M. Reick, L. C. Wu, S. L. McKnight, Science 293, 510 (2001)), anobservation consistent with the recent finding that the NAD+-dependentprotein deacetylase SIRT1 modulates activity of the clock complex (Y.Nakahata et al., Cell 134, 329 (2008); G. Asher et al., Cell 134, 317(2008)).

U.S. Pat. No. 8,106,184 describes methods of manufacturing and usingnicotinoyl riboside compositions.

U.S. application Ser. No. 11/396,359 describes nicotinamide ribosideanalogues and their uses.

U.S. application Ser. No. 11/053,185 describes methods and compositionsfor modulating the life span of eukaryotic and prokaryotic cells and forprotecting cells against certain stresses, including modulating the fluxof the NAD+ salvage pathway in the cell.

There remains a need for improved compositions and methods of using suchcompositions for pharmacologic intervention and/or manipulation of theNAD pathway in mammalian cells and tissues.

SUMMARY OF THE INVENTION

The invention relates to compositions of nicotinamide mononucleotidederivatives and their methods of use. In some embodiments, the inventionrelates to methods of making nicotinamide mononucleotide derivatives. Insome embodiments, the invention relates to pharmaceutical compositionsand nutritional supplements containing one or more nicotinamidemononucleotide derivatives. In further embodiments, the inventionrelates to methods of using nicotinamide mononucleotide derivatives thatpromote the increase of intracellular levels of nicotinamide adeninedinucleotide (NAD+) in cells and tissues for treating diseases andimproving cell and tissue survival.

DETAILED DESCRIPTION OF THE INVENTION

The advantages of the present invention include, without limitation,compounds and compositions of nicotinamide mononucleotide derivativesand their methods of use. In some embodiments, the invention relates tomethods of making nicotinamide mononucleotide derivatives. In someembodiments, the invention relates to pharmaceutical compositions andnutritional supplements containing one or more nicotinamidemononucleotide derivatives. In further embodiments, the inventionrelates to methods of using nicotinamide mononucleotide derivatives thatpromote the increase of intracellular levels of nicotinamide adeninedinucleotide (NAD+) in cells and tissues for treating diseases andimproving cell and tissue survival.

Compounds, Compositions and Methods of Treatment

Provided herein are compounds, and their stereoisomers, salts, hydrates,solvates, and crystalline forms thereof, wherein the compound has astructure represented by formula II:

wherein

-   -   (a) R¹ is hydrogen; n-alkyl; branched alkyl; cycloalkyl; or        aryl, which includes, but is not limited to, phenyl or naphthyl,        where phenyl or naphthyl are optionally substituted with at        least one of C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆        alkoxy, F, Cl, Br, I, nitro, cyano, C₁₋₆ haloalkyl, —N(R^(1′))₂,        C₁₋₆ acylamino, —NHSO₂C₁₋₆ alkyl, —SO₂N(R^(1′))₂, COR^(1″), and        —SO₂C₁₋₆ alkyl;        -   R^(1′) is independently hydrogen or alkyl, which includes,            but is not limited to, C₁₋₂₀ alkyl, C₁₋₁₀ alkyl, or C₁₋₆            alkyl; and        -   R^(1″) is —OR′ or —N(R^(1′))₂;    -   (b) R² is hydrogen; C₁₋₁₀ alkyl; or —C(O)CR^(3a)R^(3b)NHR¹,        where n is 2 to 4; or        -   R^(3a); R^(3b) and R² together are (CH₂)_(n) forming a            cyclic ring that includes the adjoining N and C atoms;    -   (c) R^(3a) and R^(3b) are        -   (i) independently selected from hydrogen, C₁₋₁₀ alkyl,            cycloalkyl, —(CH₂)_(c)(NR^(3′))₂, C₁₋₆ hydroxyalkyl, —CH₂SH,            —(CH₂)₂S(O)_(d)Me, —(CH₂)₃NHC(═NH)NH₂,            (1H-indol-3-yl)methyl, (1H-imidazol-4-yl)methyl,            —(CH₂)_(e)COR^(3″), aryl and aryl C₁₋₃ alkyl, said aryl            groups are optionally substituted with a group selected from            hydroxyl, C₁₋₁₀ alkyl, C₁₋₆ alkoxy, halogen, nitro and            cyano; or        -   (ii) R^(3a) and R^(3b) both are C₁₋₆ alkyl; or        -   (iii) R^(3a) and R^(3b) together are (CH₂)^(f) so as to form            a spiro ring; or        -   (iv) R^(3a) is hydrogen and R^(3b) and R² together are            (CH₂)_(n) so as to form a cyclic ring that includes the            adjoining N and C atoms; or        -   (v) R^(3b) is hydrogen and R^(3a) and R² together are            (CH₂)_(n) so as to form a cyclic ring that includes the            adjoining N and C atoms; or        -   (vi) R^(3a) is H and R^(3b) is H, CH₃, CH₂CH₃, CH(CH₃)₂,            CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, CH₂Ph, CH₂-indol-3-yl,            —CH₂CH₂SCH₃, CH₂CO₂H, CH₂C(O)NH₂, CH₂CH₂COOH, CH₂CH₂C(O)NH₂,            CH₂CH₂CH₂CH₂NH₂, —CH₂CH₂CH₂NHC(NH)NH₂, CH₂-imidazol-4-yl,            CH₂OH, CH(OH)CH₃, CH₂((4′-OH)-Ph), CH₂SH, or lower            cycloalkyl; or        -   (viii) R^(3a) is CH₃, —CH₂CH₃, CH(CH₃)₂, CH₂CH(CH₃)₂,            CH(CH₃)CH₂CH₃, CH₂Ph, CH₂-indol-3-yl, —CH₂CH₂SCH₃, CH₂CO₂H,            CH₂C(O)NH₂, CH₂CH₂COOH, CH₂CH₂C(O)NH₂, CH₂CH₂CH₂CH₂NH₂,            —CH₂CH₂CH₂NHC(NH)NH₂, CH₂-imidazol-4-yl, CH₂OH, CH(OH)CH₃,            CH₂((4′-OH)-Ph), CH₂SH, or lower cycloalkyl and R^(3b) is H,            where R^(3′) is independently hydrogen or alkyl, which            includes, but is not limited to, C₁₋₂₀ alkyl, C₁₋₁₀ alkyl,            or C₁₋₆ alkyl, and R^(3″) is —OR′ or —N(R^(3′))₂);        -   c is from 1 to 6,        -   d is from 0 to 2,        -   e is from 0 to 3,        -   f is from 2 to 5,        -   n is from 2 to 4, and    -   (d) R⁴ is hydrogen; C₁₋₁₀ alkyl optionally substituted with a        lower alkyl, alkoxy, di(lower alkyl)-amino, or halogen; C₁₋₁₀        haloalkyl; C₃₋₁₀ cycloalkyl; cycloalkyl alkyl; cycloheteroalkyl;        aminoacyl; aryl, such as phenyl; or heteroaryl, such as,        pyridinyl; substituted aryl; or substituted heteroaryl.

In certain embodiments, provided herein are compounds of formula II, andstereoisomers, salts, and crystalline forms thereof.

Provided herein are compounds, and their stereoisomers, salts, hydrates,solvates, and crystalline forms thereof, wherein the compound isselected from

Provided herein are compositions for the treatment and/or prophylaxis ofany of the diseases disclosed herein, comprising a pharmaceuticallyacceptable medium selected from an excipient, carrier, diluent, andequivalent medium, and a compound, or a stereoisomer, salt, hydrate,solvate, or crystalline form thereof, wherein the compound has astructure represented by formula II:

wherein

-   -   (a) R¹ is hydrogen; n-alkyl; branched alkyl; cycloalkyl; or        aryl, which includes, but is not limited to, phenyl or naphthyl,        where phenyl or naphthyl are optionally substituted with at        least one of C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆        alkoxy, F, Cl, Br, I, nitro, cyano, C₁₋₆ haloalkyl, —N(R^(1′))₂,        C₁₋₆ acylamino, —NHSO₂C₁₋₆ alkyl, —SO₂N(R^(1′))₂, COR^(1″), and        —SO₂C₁₋₆ alkyl;        -   R^(1′) is independently hydrogen or alkyl, which includes,            but is not limited to, C₁₋₂₀ alkyl, C₁₋₁₀ alkyl, or C₁₋₆            alkyl; and        -   R^(1″) is —OR′ or —N(R^(1′))₂;    -   (b) R² is hydrogen; C₁₋₁₀ alkyl; or —C(O)CR^(3a)R^(3b)NHR¹,        where n is 2 to 4; or        -   R^(3a); R^(3b) and R² together are (CH₂)_(n) forming a            cyclic ring that includes the adjoining N and C atoms;    -   (c) R^(3a) and R^(3b) are        -   (i) independently selected from hydrogen, C₁₋₁₀ alkyl,            cycloalkyl, —(CH₂)(NR^(3′))₂, C₁₋₆ hydroxyalkyl, —CH₂SH,            —(CH₂)₂S(O)_(d)Me, —(CH₂)₃NHC(═NH)NH₂,            (1H-indol-3-yl)methyl, (1H-imidazol-4-yl)methyl,            —(CH₂)_(e)COR^(3″), aryl and aryl C₁₋₃ alkyl, said aryl            groups are optionally substituted with a group selected from            hydroxyl, C₁₋₁₀ alkyl, C₁₋₆ alkoxy, halogen, nitro and            cyano; or        -   (ii) R^(3a) and R^(3b) both are C₁₋₆ alkyl; or        -   (iii) R^(3a) and R^(3b) together are (CH₂)_(f) so as to form            a spiro ring; or        -   (iv) R^(3a) is hydrogen and R^(3b) and R² together are            (CH₂)_(n) so as to form a cyclic ring that includes the            adjoining N and C atoms; or        -   (v) R^(3b) is hydrogen and R^(3a) and R² together are            (CH₂)_(n) so as to form a cyclic ring that includes the            adjoining N and C atoms; or        -   (vi) R^(3a) is H and R^(3b) is H, CH₃, CH₂CH₃, CH(CH₃)₂,            CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, CH₂Ph, CH₂-indol-3-yl,            —CH₂CH₂SCH₃, CH₂CO₂H, CH₂C(O)NH₂, CH₂CH₂COOH, CH₂CH₂C(O)NH₂,            CH₂CH₂CH₂CH₂NH₂, —CH₂CH₂CH₂NHC(NH)NH₂, CH₂-imidazol-4-yl,            CH₂OH, CH(OH)CH₃, CH₂((4′-OH)-Ph), CH₂SH, or lower            cycloalkyl; or        -   (viii) R^(3a) is CH₃, —CH₂CH₃, CH(CH₃)₂, CH₂CH(CH₃)₂,            CH(CH₃)CH₂CH₃, CH₂Ph, CH₂-indol-3-yl, —CH₂CH₂SCH₃, CH₂CO₂H,            CH₂C(O)NH₂, CH₂CH₂COOH, CH₂CH₂C(O)NH₂, CH₂CH₂CH₂CH₂NH₂,            —CH₂CH₂CH₂NHC(NH)NH₂, CH₂-imidazol-4-yl, CH₂OH, CH(OH)CH₃,            CH₂((4′-OH)-Ph), CH₂SH, or lower cycloalkyl and R^(3b) is H,            where R^(3′) is independently hydrogen or alkyl, which            includes, but is not limited to, C₁₋₂₀ alkyl, C₁₋₁₀ alkyl,            or C₁₋₆ alkyl, and R^(3″) is —OR′ or —N(R^(3′))₂);        -   c is from 1 to 6,        -   d is from 0 to 2,        -   e is from 0 to 3,        -   f is from 2 to 5,        -   n is from 2 to 4, and    -   (d) R⁴ is hydrogen; C₁₋₁₀ alkyl optionally substituted with a        lower alkyl, alkoxy, di(lower alkyl)-amino, or halogen; C₁₋₁₀        haloalkyl; C₃₋₁₀ cycloalkyl; cycloalkyl alkyl; cycloheteroalkyl;        aminoacyl; aryl, such as phenyl; or heteroaryl, such as,        pyridinyl; substituted aryl; or substituted heteroaryl.

Provided herein are methods of treating a disease or disorder associatedwith NAD+ biosynthesis, comprising administering a compound, or astereoisomer, salt, hydrate, solvate, or crystalline form thereof, to asubject in need thereof; wherein the compound has a structurerepresented by formula II:

wherein

-   -   (a) R¹ is hydrogen, n-alkyl; branched alkyl; cycloalkyl; or        aryl, which includes, but is not limited to, phenyl or naphthyl,        where phenyl or naphthyl are optionally substituted with at        least one of C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆        alkoxy, F, Cl, Br, I, nitro, cyano, C₁₋₆ haloalkyl, —N(R^(1′))₂,        C₁₋₆ acylamino, —NHSO₂C₁₋₆ alkyl, —SO₂N(R^(1′))₂, COR^(1″), and        —SO₂C₁₋₆ alkyl;        -   R^(1′) is independently hydrogen or alkyl, which includes,            but is not limited to, C₁₋₂₀ alkyl, C₁₋₁₀ alkyl, or C₁₋₆            alkyl, and        -   R^(1″) is —OR′ or —N(R^(1′))₂;    -   (b) R² is hydrogen, C₁₋₁₀ alkyl; or C(O)CR^(3a)R^(3b)NHR¹, where        n is 2 to 4; or        -   R^(3a) or R^(3b) and R² together are (CH₂)_(n) forming a            cyclic ring that includes the adjoining N and C atoms;    -   (c) R^(3a) and R^(3b) are        -   (i) independently selected from hydrogen, C₁₋₁₀ alkyl,            cycloalkyl, —(CH₂)_(c)(NR³)₂, C₁₋₆ hydroxyalkyl, —CH₂SH,            —(CH₂)₂S(O)_(d)Me, —(CH₂)₃NHC(═NH)NH₂,            (1H-indol-3-yl)methyl, (1H-imidazol-4-yl)methyl,            (CH₂)_(e)COR^(3″), aryl and aryl C₁₋₃ alkyl, said aryl            groups optionally substituted with a group selected from            hydroxyl, C₁₋₁₀ alkyl, C₁₋₆ alkoxy, halogen, nitro and            cyano;        -   (ii) R^(3a) and R^(3b) both are C₁₋₆ alkyl;        -   (iii) R^(3a) and R^(3b) together are (CH₂)_(f) so as to form            a spiro ring;        -   (iv) R^(3a) is hydrogen and R^(3b) and R² together are            (CH₂)_(n) forming a cyclic ring that includes the adjoining            N and C atoms;        -   (v) R^(3b) is hydrogen and R^(3a) and R² together are            (CH₂)_(n) forming a cyclic ring that includes the adjoining            N and C atoms, where, and where R^(3′) is independently            hydrogen or C₁₋₆ alkyl and R^(3″) is —OR′ or —N(R^(3′))₂);        -   (vi) R^(3a) is H and R^(3b) is H, CH₃, CH₂CH₃, CH(CH₃)₂,            CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, CH₂Ph, CH₂-indol-3-yl,            CH₂CH₂SCH₃, CH₂CO₂H, CH₂C(O)NH₂, CH₂CH₂COOH, CH₂CH₂C(O)NH₂,            CH₂CH₂CH₂CH₂NH₂, CH₂CH₂CH₂NHC(NH)NH₂, CH₂-imidazol-4-yl,            CH₂OH, CH(OH)CH₃, CH₂((4′-OH)-Ph), CH₂SH, or lower            cycloalkyl; or        -   (vii) R^(3a) is CH₃, CH₂CH₃, CH(CH₃)₂, CH₂CH(CH₃)₂,            CH(CH₃)CH₂CH₃, CH₂Ph, CH₂-indol-3-yl, CH₂CH₂SCH₃, CH₂CO₂H,            CH₂C(O)NH₂, CH₂CH₂COOH, CH₂CH₂C(O)NH₂, CH₂CH₂CH₂CH₂NH₂,            CH₂CH₂CH₂NHC(NH)NH₂, CH₂-imidazol-4-yl, CH₂OH, CH(OH)CH₃,            CH₂((4′-OH)-Ph), CH₂SH, or lower cycloalkyl and R^(3b) is H;            and        -   c is from 1 to 6,        -   d is from 0 to 2,        -   e is from 0 to 3,        -   f is from 2 to 5,        -   n is from 2 to 4, and    -   (d) R⁴ is hydrogen, C₁₋₁₀ alkyl, C₁₋₁₀ alkyl optionally        substituted with lower alkyl, alkoxy, di(lower alkyl)-amino, or        halogen, C₁₋₁₀ haloalkyl, C₃₋₁₀ cycloalkyl, cycloalkyl alkyl,        cycloheteroalkyl, aminoacyl, aryl, such as phenyl, heteroaryl,        such as, pyridinyl, substituted aryl, or substituted heteroaryl.

Provided herein are methods of treatment as disclosed above and herein,wherein the compound is selected from

Provided herein are compounds, and their stereoisomers, salts, hydrates,solvates, and crystalline forms thereof, wherein the compound has astructure represented by formula III:

wherein each W¹ and W² is independently

-   -   (i) each R^(c) and R^(d) is independently H, (C₁-C₅)alkyl,        (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₃-C₈)carbocyclyl,        (C₄-C₈)carbocyclylalkyl, aryl(C₁-C₈)alkyl,        heterocyclyl(C₁-C₈)alkyl, (C₆-C₂₀)aryl, (C₂-C₂₀)heterocyclyl or        heteroaryl; or    -   (ii) each R^(c) is H and each R^(d) is independently        (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,        (C₃-C₈)carbocyclyl, (C₄-C₈)carbocyclylalkyl, aryl(C₁-C₈)alkyl,        heterocyclyl(C₁-C₈)alkyl, (C₆-C₂₀)aryl, (C₂-C₂₀)heterocyclyl or        heteroaryl; or    -   (iii) each R^(c) is H and each R^(d) is independently        (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,        (C₃-C₈)carbocyclyl, (C₄-C₈)carbocyclylalkyl, aryl(C₁-C₈)alkyl,        heterocyclyl(C₁-C₈)alkyl, (C₆-C₂₀)aryl, (C₂-C₂₀)heterocyclyl or        heteroaryl wherein the chirality of the carbon to which said        R^(c) and R^(d) is attached is S; or    -   (iv) each R^(c) is H and each R^(d) is independently        (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,        (C₃-C₈)carbocyclyl, (C₄-C₈)carbocyclylalkyl, aryl(C₁-C₈)alkyl,        heterocyclyl(C₁-C₈)alkyl, (C₆-C₂₀)aryl, (C₂-C₂₀)heterocyclyl or        heteroaryl wherein the chirality of the carbon to which said        R^(c) and R^(d) is attached is R; or    -   (v) each R^(c) is H and each R^(d) is independently        (C₁-C₈)alkyl; or    -   (vi) each R^(c) is H and each R^(d) is independently        (C₁-C₈)alkyl wherein the chirality of the carbon to which said        R^(c) and R^(d) is attached is S; or    -   (vii) each R^(c) is H and each R^(d) is independently        (C₁-C₈)alkyl wherein the chirality of the carbon to which said        R^(c) and R^(d) is attached is R; and        each R⁶ is independently (C₁-C₈)alkyl, (C₂-C₈)alkenyl,        (C₂-C₈)alkynyl, (C₃-C₈)carbocyclyl or (C₄-C₈)carbocyclylalkyl.

In certain embodiments of Formula III, each

comprises a nitrogen-linked naturally occurring α-amino acid ester.

In some embodiments of Formula III, each W¹ and W² is independently

and each R⁶ is independently (C₁-C₈)alkyl.

In certain embodiments, each R^(c) and R^(d) is independently H,(C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₃-C₈)carbocyclyl,(C₄-C₈)carbocyclylalkyl, aryl(C₁-C₈)alkyl, heterocyclyl(C₁-C₈)alkyl,(C₆-C₂₀)aryl, (C₂-C₂₀)heterocyclyl or heteroaryl. In certainembodiments, each R^(c) is H and each R^(d) is independently(C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₃-C₈)carbocyclyl,(C₄-C₈)carbocyclylalkyl, aryl(C₁-C₈)alkyl, heterocyclyl(C₁-C₈)alkyl,(C₆-C₂₀)aryl, (C₂-C₂₀)heterocyclyl or heteroaryl. In certainembodiments, each R^(c) is H and each R^(d) is independently(C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₃-C₈)carbocyclyl,(C₄-C₈)carbocyclylalkyl, aryl(C₁-C₈)alkyl, heterocyclyl(C₁-C₈)alkyl,(C₆-C₂₀)aryl, (C₂-C₂₀)heterocyclyl or heteroaryl, wherein the chiralityof the carbon to which said R^(c) and R^(d) is attached is S. In certainembodiments, each R^(c) is H and each R^(d) is independently(C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₃-C₈)carbocyclyl,(C₄-C₈)carbocyclylalkyl, aryl(C₁-C₈)alkyl, heterocyclyl(C₁-C₈)alkyl,(C₆-C₂₀)aryl, (C₂-C₂₀)heterocyclyl or heteroaryl, wherein the chiralityof the carbon to which said R^(c) and R^(d) is attached is R. In certainembodiments, each R^(c) is H and each R^(d) is independently(C₁-C₈)alkyl. In certain embodiments, each R^(c) is H and each R^(d) isindependently (C₁-C₈)alkyl, wherein the chirality of the carbon to whichsaid R^(c) and R^(d) is attached is S. In certain embodiments, eachR^(c) is H and each R^(d) is independently (C₁-C₈)alkyl, wherein thechirality of the carbon to which said R^(c) and R^(d) is attached is R.

In certain embodiments, each

comprises a nitrogen-linked naturally occurring α-amino acid ester.

In some embodiments of Formula III, each W¹ and W² is independently

and each R⁶ is independently (C₁-C₈)alkyl.

In certain embodiments, each R⁶ is independently secondary alkyl. Incertain embodiments, each R⁶ is 2-propyl. In certain embodiments, eachR^(c) is H and each R^(d) is methyl. In certain embodiments, each R^(c)is H and each R^(d) is methyl, wherein the chirality of the carbon towhich said R^(c) and R^(d) is attached is S. In certain embodiments,each R^(c) is H and each R^(d) is methyl, wherein the chirality of thecarbon to which said R^(c) and R^(d) is attached is R.

In certain embodiments, each

comprises a nitrogen-linked naturally occurring α-amino acid ester.

In some embodiments of Formula III, one of W¹ or W² is OR⁵ and the otherof W¹ or W² is

In certain embodiments, R⁵ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, (C₃-C₈)carbocyclyl, (C₄-C₈)carbocyclylalkyl,aryl(C₁-C₈)alkyl, heterocyclyl(C₁-C₈)alkyl, (C₆-C₂₀)aryl,(C₂-C₂₀)heterocyclyl or heteroaryl. In certain embodiments, R⁵ is(C₁-C₈)alkyl. In certain embodiments, R⁵ is (C₆-C₂₀)aryl,(C₂-C₂₀)heterocyclyl or heteroaryl. In certain embodiments, R⁵ is(C₆-C₂₀)aryl. In certain embodiments, R⁵ is phenyl.

In certain embodiments, each

comprises a nitrogen-linked naturally occurring α-amino acid ester.

In some embodiments of Formula III, one of W¹ or W² is OR⁵ and the otherof W¹ or W² is

wherein R⁵ is unsubstituted phenyl.

In certain embodiments, each R^(c) and R^(d) is independently H,(C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₃-C₈)carbocyclyl,(C₄-C₈)carbocyclylalkyl, aryl(C₁-C₈)alkyl, heterocyclyl(C₁-C₈)alkyl,(C₆-C₂₀)aryl, (C₂-C₂₀)heterocyclyl or heteroaryl. In certainembodiments, one of R^(c) or R^(d) is H and the other of R^(c) or R^(d)is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₃-C₈)carbocyclyl,(C₄-C₈)carbocyclylalkyl, aryl(C₁-C₈)alkyl, heterocyclyl(C₁-C₈)alkyl,(C₆-C₂₀)aryl, (C₂-C₂₀)heterocyclyl or heteroaryl. In certainembodiments, one of R^(c) or R^(d) is H and the other of R^(c) or R^(d)is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₃-C₈)carbocyclyl,(C₄-C₈)carbocyclylalkyl, aryl(C₁-C₈)alkyl, heterocyclyl(C₁-C₈)alkyl,(C₆-C₂₀)aryl, (C₂-C₂₀)heterocyclyl or heteroaryl, wherein the chiralityof the carbon to which said R^(c) and R^(d) is attached is S. In certainembodiments, one of R^(c) or R^(d) is H and the other of R^(c) or R^(d)is independently (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₃-C₈)carbocyclyl, (C₄-C₈)carbocyclylalkyl, aryl(C₁-C₈)alkyl,heterocyclyl(C₁-C₈)alkyl, (C₆-C₂₀)aryl, (C₂-C₂₀)heterocyclyl orheteroaryl, wherein the chirality of the carbon to which said R^(c) andR^(d) is attached is R. In certain embodiments, one of R^(c) or R^(d) isH and the other of R^(c) or R^(d) is (C₁-C₈)alkyl. In certainembodiments, one of R^(c) or R^(d) is H and the other of R^(c) or R^(d)is (C₁-C₈)alkyl, wherein the chirality of the carbon to which said R^(c)and R^(d) is attached is S. In certain embodiments, one of R^(c) orR^(d) is H and the other of R^(c) or R^(d) is (C₁-C₈)alkyl, wherein thechirality of the carbon to which said R^(c) and R^(d) is attached is R.In certain embodiments, the chirality at phosphorus is S. In certainembodiments, the chirality at phosphorus is R.

In certain embodiments, each

comprises a nitrogen-linked naturally occurring α-amino acid ester.

In some embodiments of Formula III, one of W¹ or W² is OR⁵ and the otherof W or W² is

wherein one of R^(c) or R^(d) is H and the other of R^(c) or R^(d) ismethyl.

In certain embodiments, one of R^(c) or R^(d) is H and the other ofR^(c) or R^(d) is methyl, wherein the chirality of the carbon to whichsaid R^(c) and R^(d) is attached is S. In certain embodiments, one ofR^(c) or R^(d) is H and the other of R^(c) or R^(d) is methyl, whereinthe chirality of the carbon to which said R^(c) and R^(d) is attached isR. In certain embodiments, R⁵ is phenyl.

In certain embodiments, each

comprises a nitrogen-linked naturally occurring α-amino acid ester.

In some embodiments of Formula III, one of W¹ or W² is OR⁵ and the otherof W¹ or W² is

wherein R⁵ is unsubstituted phenyl, one of R^(c) or R^(d) is H and theother of R^(c) or R^(d) is methyl.

In certain embodiments, the chirality at phosphorous is R. In certainembodiments, the chirality at phosphorous is S. In certain embodimentsthe chirality of the carbon to which said R^(c) and R^(d) is attached isS. In certain embodiments, the chirality of the carbon to which saidR^(c) and R^(d) is attached is S and the chirality at phosphorus is S.In certain embodiments, the chirality of the carbon to which said R^(c)and R^(d) is attached is S and the chirality at phosphorus is R. Incertain embodiments, the chirality of the carbon to which said R^(c) andR^(d) is attached is R. In certain embodiments, the chirality of thecarbon to which said R^(c) and R^(d) is attached is R and the chiralityat phosphorus is S. In certain embodiments, the chirality of the carbonto which said R^(c) and R^(d) is attached is R and the chirality atphosphorus is R.

In certain embodiments, each

comprises a nitrogen-linked naturally occurring α-amino acid ester.

In some embodiments of Formula III, one of W¹ or W² is OR⁵ and the otherof W¹ or W² is

wherein R⁵ is unsubstituted phenyl and R⁶ is (C₁-C₈)alkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₃-C₈)carbocyclyl, or(C₄-C₈)carbocyclylalkyl.

In certain embodiments, R⁶ is (C₁-C₈)alkyl. In certain embodiments, oneof R^(c) or R^(d) is H and the other of R^(c) or R^(d) is (C₁-C₈)alkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₃-C₈)carbocyclyl,(C₄-C₈)carbocyclylalkyl, aryl(C₁-C₈)alkyl, heterocyclyl(C₁-C₈)alkyl,(C₆-C₂₀)aryl, (C₂-C₂₀)heterocyclyl or heteroaryl. In certainembodiments, one of R^(c) or R^(d) is H and the other of R^(c) or R^(d)is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₃-C₈)carbocyclyl,(C₄-C₈)carbocyclylalkyl, aryl(C₁-C₈)alkyl, heterocyclyl(C₁-C₈)alkyl,(C₆-C₂₀)aryl, (C₂-C₂₀)heterocyclyl or heteroaryl, wherein the chiralityof the carbon to which said R^(c) and R^(d) is attached is S. In certainembodiments, one of R^(c) or R^(d) is H and the other of R^(c) or R^(d)is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₃-C₈)carbocyclyl,(C₄-C₈)carbocyclylalkyl, aryl(C₁-C₈)alkyl, heterocyclyl(C₁-C₈)alkyl,(C₆-C₂₀)aryl, (C₂-C₂₀)heterocyclyl or heteroaryl, wherein the chiralityof the carbon to which said R^(c) and R^(d) is attached is R. In certainembodiments, one of R^(c) or R^(d) is H and the other of R^(c) or R^(d)is (C₁-C₈)alkyl. In certain embodiments, one of R^(c) or R^(d) is H andthe other of R^(c) or R^(d) is (C₁-C₈)alkyl, wherein the chirality ofthe carbon to which said R^(c) and R^(d) is attached is S. In certainembodiments, one of R^(c) or R^(d) is H and the other of R^(c) or R^(d)is (C₁-C₈)alkyl, wherein the chirality of the carbon to which said R^(c)and R^(d) is attached is R. In certain embodiments, the chirality atphosphorus is S. In certain embodiments, the chirality at phosphorus isR.

In certain embodiments, each

comprises a nitrogen-linked naturally occurring α-amino acid ester.

In some embodiments of Formula III, one of W¹ or W² is OR⁵ and the otherof W¹ or W² is

wherein one of R^(c) or R^(d) is H and the other of R^(c) or R^(d) ismethyl and R⁶ is (C₁-C₈)alkyl.

In certain embodiments, R⁶ is secondary alkyl. In certain embodiments,R⁶ is 2-propyl. In certain embodiments, the chirality of the carbon towhich said R^(c) and R^(d) is attached is S. In certain embodiments, thechirality of the carbon to which said R^(c) and R^(d) is attached is R.In certain embodiments, R⁵ is phenyl.

In certain embodiments, each

comprises a nitrogen-linked naturally occurring α-amino acid ester.

In some embodiments of Formula III, one of W¹ or W² is OR⁵ and the otherof W¹ or W² is

wherein R⁵ is unsubstituted phenyl, one of R^(c) or R^(d) is H and theother of R^(c) or R^(d) is methyl and R⁶ is (C₁-C₈)alkyl.

In certain embodiments, R⁶ is secondary alkyl. In certain embodiments,R⁶ is 2-propyl. In certain embodiments, the chirality of the carbon towhich said R^(c) and R^(d) is attached is S. In certain embodiments, thechirality of the carbon to which said R^(c) and R^(d) is attached is R.

In certain embodiments, each

comprises a nitrogen-linked naturally occurring α-amino acid ester.

In certain embodiments, each R^(c) is H and each R^(d) is methyl. Incertain embodiments, each R^(c) is H and each R^(d) is methyl, whereinthe chirality of the carbon to which each said R^(c) and R^(d) isattached is S. In certain embodiments, each R^(c) is H and each R^(d) ismethyl, wherein the chirality of the carbon to which each said R^(c) andR^(d) is attached is R. In certain embodiments, R⁵ is phenyl.

In certain embodiments, each R⁶ is independently (C₁-C₈)alkyl. Incertain embodiments, each R⁶ is independently secondary alkyl. Incertain embodiments, each W¹ and W² is independently

In some embodiments, one of W¹ or W² is OR⁵ and the other of W¹ or W² is

In some embodiments, each

comprises a nitrogen-linked naturally occurring α-amino acid ester.

In certain embodiments, one of R^(c) or R^(d) is H and the other ofR^(c) or R^(d) is methyl. In certain embodiments, one of R^(c) or R^(d)is H and the other of R^(c) or R^(d) is methyl, wherein the chirality ofthe carbon to which said R^(c) and R^(d) is attached is S. In certainembodiments, one of R^(c) or R^(d) is H and the other of R^(c) or R^(d)is methyl, wherein the chirality of the carbon to which said R^(c) andR^(d) is attached is R. In certain embodiments, R⁵ is phenyl. In certainembodiments, the chirality at phosphorus is S. In certain embodiments,the chirality at phosphorus is R.

In certain embodiments, each

comprises a nitrogen-linked naturally occurring α-amino acid ester.

In certain embodiments, R⁶ is (C₁-C₈)alkyl. In certain embodiments, R⁶is secondary alkyl. In certain embodiments, one of R^(c) or R^(d) is Hand the other of R^(c) or R^(d) is methyl. In certain embodiments, oneof R^(c) or R^(d) is H and the other of R^(c) or R^(d) is methyl,wherein the chirality of the carbon to which said R^(c) and R^(d) isattached is S. In certain embodiments, one of R^(c) or R^(d) is H andthe other of R^(c) or R^(d) is methyl, wherein the chirality of thecarbon to which said R^(c) and R^(d) is attached is R. In certainembodiments, R⁵ is phenyl. In certain embodiments, the chirality atphosphorus is S. In certain embodiments, the chirality at phosphorus isR.

In certain embodiments, each

comprises a nitrogen-linked naturally occurring α-amino acid ester.

Provided herein are compounds, and their stereoisomers, salts, hydrates,solvates, and crystalline forms thereof, wherein the compound has astructure represented by formula III:

wherein W¹ and W² are each independently O⁻ or OR⁵, and R⁵ is hydrogenor alkyl; provided that when W¹ is O⁻, and W² is OR⁵, then R⁵ is nothydrogen, methyl or butyl.

In certain embodiments, R⁵ is (C₁-C₈)alkyl. In certain embodiments, R⁵is selected from methyl, ethyl, n-propyl, isopropyl, and butyl. Incertain embodiments, one R⁵ is hydrogen and the other R⁵ is methyl. Incertain embodiments, one R⁵ is hydrogen and the other R⁵ is methyl. Incertain embodiments, one R⁵ is O⁻ and the other R⁵ is C₂, C₃, orC₅-C₈-alkyl. In certain embodiments, both R⁵ are (C₁-C₈)alkyl.

Provided herein are compounds, and their stereoisomers, salts, hydrates,solvates, and crystalline forms thereof, wherein the compound has astructure represented by formula III:

wherein W¹ and W² are independently selected from the substituents inTable 1. Synthesis and general descriptions of representativesubstituents can be found, for instance, in U.S. Pat. No. 8,318,682,incorporated herein by reference in its entirety. The variables used inTable 1 (e.g., W²³, R²¹, etc.) pertain only to Table 1, unless otherwiseindicated.

The variables used in Table 1 have the following definitions:

-   -   each R²¹ is independently H or (C₁-C₈)alkyl;    -   each R²² is independently H, R²¹, R²³ or R²⁴, wherein each R²⁴        is independently substituted with 0 to 3 R²³;    -   each R²³ is independently R^(23a), R^(23b), R^(23c) or R^(23d),        provided that when R²³ is bound to a heteroatom, then R²³ is        R^(23c) or R^(23d);    -   each R^(23a) is independently F, Cl, Br, I, —CN, —N₃ or —NO₂;    -   each R^(23b) is independently Y²¹;    -   each R^(23c) is independently —R^(2x), —OR^(2x),        —N(R^(2x))(R^(2x)), —SR^(2x), —S(O)R^(2x); —S(O)₂R^(2x),        —S(O)(OR^(2x)), —S(O)₂(OR^(2x)), —OC(═Y²¹)R^(2x),        —OC(═Y²¹)OR^(2x), —OC(═Y²¹)(N(R^(2x))(R^(2x))); —SC(═Y²¹)R^(2x);        —SC(═Y²¹)OR^(2x), —SC(═Y²¹)(N(R²)(R^(2x))),        —N(R^(2x))C(═Y²¹)R^(2x), —N(R^(2x))C(═Y²¹)OR^(2x), or        —N(R^(2x))C(═Y²¹)(N(R^(2x))(R^(2x)));    -   each R^(23d) is independently —C(═Y²¹)R^(2x); —C(═Y²¹)OR^(2x) or        —C(═Y²¹)(N(R^(2x))(R^(2x)));    -   each R^(2x) is independently H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl,        (C₂-C₈)alkynyl, aryl, heteroaryl; or two R^(2x) taken together        with a nitrogen or oxygen to which they are both attached form a        3 to 7 membered heterocyclic ring wherein any one carbon atom of        said heterocyclic ring can optionally be replaced with —O—, —S—        or —NR²¹—; and wherein one or more of the non-terminal carbon        atoms of each said (C₁-C₈)alkyl may be optionally replaced with        —O—, —S— or —NR²¹—;    -   each R²⁴ is independently (C₁-C₈)alkyl, (C₂-C₈)alkenyl, or        (C₂-C₈)alkynyl;    -   each R²⁵ is independently R²⁴, wherein each R²⁴ is substituted        with 0 to 3 R²³ groups;    -   each R^(25a) is independently (C₁-C₈)alkylene,        (C₂-C₈)alkenylene, or (C₂-C₈)alkynylene, any one of which said        (C₁-C₈)alkylene, (C₂-C₈)alkenylene, or (C₂-C₈)alkynylene is        substituted with 0-3 R²³ groups;    -   each W²³ is independently W²⁴ or W²⁵;    -   each W²⁴ is independently R²⁵, —C(═Y²¹)R²⁵, —C(═Y²¹)W²⁵,        —SO₂R²⁵, —SO₂W²⁵;    -   each W²⁵ is independently carbocycle or heterocycle wherein W²⁵        is independently substituted with 0 to 3 R²² groups; and    -   each Y²¹ is independently O or S.

TABLE 1 W¹ and W² Substituents

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99

100

101

102

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180

181

182

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

198

199

200

201

202

203

204

205

206

207

208

209

210

211

212

213

214

215

216

217

218

219

220

221

222

223

224

225

226

227

228

229

230

231

232

233

234

225

226

227

228

229

230

231

232

233

234

225

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

247

248

249

250

251

252

253

254

255

256

257

258

103

104

105

In certain embodiments, provided herein are compounds of formula III,and their stereoisomers, salts, and crystalline forms thereof.

Provided herein are methods of treating a disease or disorder associatedwith NAD+ biosynthesis, comprising administering a compound, or itsstereoisomer, salt, hydrate, solvate, or crystalline form thereof, to asubject in need thereof; wherein the compound is represented by formulaIII:

wherein W¹ and W² are independently selected from the substituents inTable 2.

Provided herein are methods of treatment comprising administering acompound, or a stereoisomer, salt, hydrate, solvate, or crystalline formthereof, wherein the compound is

Provided herein are compounds, and their stereoisomers, salts, hydrates,solvates, or crystalline forms thereof, wherein the compound has astructure represented by formula I:

wherein V is selected from hydrogen, phenyl and monocyclic heteroaryl,wherein (i) each said monocyclic heteroaryl contains five or six ringatoms of which 1 or 2 ring atoms are heteroatoms selected from N, S, andO, and the remainder of the ring atoms are carbon, and (ii) each saidphenyl or monocyclic heteroaryl is unsubstituted or is substituted byone or two groups selected from halogen, trifluoromethyl, C₁-C₆ alkyl,C₁-C₆ alkoxy, and cyano.

In certain embodiments, provided herein are compounds of formula I, andtheir stereoisomers, salts, or crystalline forms thereof.

Provided herein are compounds, and their stereoisomers, salts, hydrates,solvates, or crystalline forms thereof, wherein the compound has astructure represented by formula I:

wherein V is selected from the substituents in Table 2. Synthesis andgeneral descriptions of representative substituents can be found, forinstance, in U.S. Pat. No. 8,063,025, incorporated herein by referencein its entirety.

TABLE 2 V Substituents

In other embodiments, the compound is selected from:

Provided herein are compositions for the treatment and/or prophylaxis ofany of the diseases disclosed herein, said compositions comprising apharmaceutically acceptable medium selected from an excipient, carrier,diluent, and equivalent medium and a compound, or a stereoisomer, salt,hydrate, solvate, or crystalline form thereof, wherein the compound hasa structure represented by formula I:

wherein V is selected from hydrogen, phenyl and monocyclic heteroaryl,wherein (i) each said monocyclic heteroaryl contains five or six ringatoms of which 1 or 2 ring atoms are heteroatoms selected from N, S, andO, and the remainder of the ring atoms are carbon, and (ii) each saidphenyl or monocyclic heteroaryl is unsubstituted or is substituted byone or two groups selected from halogen, trifluoromethyl, C₁-C₆ alkyl,C₁-C₆ alkoxy, and cyano.

Provided herein are compositions for the treatment and/or prophylaxis ofany of the diseases disclosed herein, said compositions comprising apharmaceutically acceptable medium selected from an excipient, carrier,diluent, and equivalent medium and a compound, or a stereoisomer, salt,hydrate, solvate, or crystalline form thereof, wherein the compound hasa structure represented by formula I:

wherein V is selected from the substituents in Table 2.

Provided herein are methods of treating a disease or disorder associatedwith NAD+ biosynthesis, comprising administering a compound, or astereoisomer, salt, hydrate, solvate, or crystalline form thereof, to asubject in need thereof, wherein the compound has a structurerepresented by formula I:

wherein V is selected from hydrogen, phenyl and monocyclic heteroaryl,wherein (i) each said monocyclic heteroaryl contains five or six ringatoms of which 1 or 2 ring atoms are heteroatoms selected from N, S, andO, and the remainder of the ring atoms are carbon, and (ii) each saidphenyl or monocyclic heteroaryl is unsubstituted or is substituted byone or two groups selected from halogen, trifluoromethyl, C₁-C₆ alkyl,C₁-C₆ alkoxy, and cyano.

Provided herein are methods of treatment in a subject in need thereof,comprising administering a therapeutically effective amount of acompound, or a stereoisomer, salt, hydrate, solvate, or crystalline formthereof, to the subject; wherein the compound has a structurerepresented by formula I:

wherein V is selected from the substituents in Table 2.

Provided herein is a compound, its stereoisomer, salt, hydrate, solvate,or crystalline form thereof, wherein the compound is selected from

Provided herein is a compound, or a stereoisomer, salt, hydrate,solvate, or crystalline form thereof, wherein the compound is selectedfrom Compounds 1, 2, 11, and 12. Also provided herein is a compound, ora stereoisomer, salt, hydrate, solvate, or crystalline form thereof,wherein the compound is selected from Compounds 3, 17, 18, 19, 20, 21,and 22. Also provided herein is a compound, or a stereoisomer, salt,hydrate, solvate, or crystalline form thereof, wherein the compound isselected from Compounds 4, 5, 6, 7, 8, 9, and 10. Also provided hereinis a compound, or a stereoisomer, salt, hydrate, solvate, or crystallineform thereof, wherein the compound is selected from Compounds 13, 14,and 15.

In some embodiments, the disclosed compounds are in the form of apositively charged pyridium cation, which may form a salt with anysuitable anion. The anion can alter as the compound is isolated ortransferred into media with different anionic species. For example, adisclosed compound may be in the form a pyridium salt that is apharmaceutically acceptable salt as described herein. In certainembodiments, the pyridium compound is isolated as a salt with an anionselected from acetate, triflate, halide, trifluoroacetate, or formate.In other embodiments, if the disclosed compound is in contact with amedia, e.g., aqueous media, the anion can be selected from, for example,OH⁻, H₂PO₄ ⁻, HPO₄ ²⁻HSO₄ ⁻, SO₄ ²⁻, NO₃ ⁻HCO₃ ⁻, and CO₃ ²⁻.

Synthetic schemes for preparing compounds of Formula I, Formula II andFormula III can be found, for instance, in the following referencesincorporated herein by reference in their entirety. Nicotinamideriboside and intermediates of nicotinamide riboside with protectedfunctionalities and well-established leaving groups that could be usedin the synthesis of compounds of the present invention are described in,for example, Milburn et al. (US2006/0229265) as well as Sauve et al(U.S. Pat. No. 8,106,184). Synthetic schemes and characterization ofintermediates necessary for compounds of Formula I can be found, forinstance, in Heckler et al. (U.S. Pat. No. 8,063,025); Heckler et al.(U.S. application Ser. No. 12/745,419); Butler et al. (U.S. Pat. No.8,318,682); Cho et al. (U.S. Pat. No. 8,415,308); Ross et al (U.S.application Ser. No. 13/732,725); and Ross et al (U.S. application Ser.No. 13/076,842). Protecting groups and/or leaving groups useful forsynthesis of the compounds of the present invention can be found, forinstance, in Ross et al. (U.S. application Ser. No. 13/076,842).

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art ofthe present disclosure. The following references provide one of skillwith a general definition of many of the terms used in this disclosure:Singleton et al., Dictionary of Microbiology and Molecular Biology (2nded. 1994); The Cambridge Dictionary of Science and Technology (Walkered., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.),Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionaryof Biology (1991). As used herein, the following terms have the meaningsascribed to them below, unless specified otherwise.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

The phrase “a” or “an” entity as used herein refers to one or more ofthat entity; for example, a compound refers to one or more compounds orat least one compound. As such, the terms “a” (or “an”), “one or more”,and “at least one” can be used interchangeably herein.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

The terms “optional” or “optionally” as used herein means that asubsequently described event or circumstance may but need not occur, andthat the description includes instances where the event or circumstanceoccurs and instances in which it does not. For example, “optional bond”means that the bond may or may not be present, and that the descriptionincludes single, double, or triple bonds.

The term “P*” means that the phosphorus atom is chiral and that it has acorresponding Cahn-Ingold-Prelog designation of “R” or “S” which havetheir accepted plain meanings.

The term “purified,” as described herein, refers to the purity of agiven compound. For example, a compound is “purified” when the givencompound is a major component of the composition, i.e., at least about50% w/w pure. Thus, “purified” embraces at least about 50% w/w purity,at least about 60% w/w purity, at least about 70% purity, at least about80% purity, at least about 85% purity, at least about 90% purity, atleast about 92% purity, at least about 94% purity, at least about 96%purity, at least about 97% purity, at least about 98% purity, at leastabout 99% purity, at least about 99.5% purity, and at least about 99.9%purity, wherein “substantially pure” embraces at least about 97% purity,at least about 98% purity, at least about 99% purity, at least about99.5% purity, and at least about 99.9% purity.

The term “metabolite,” as described herein, refers to a compoundproduced in vivo after administration to a subject in need thereof.

The term “substantially anhydrous” means that a substance contains atmost 10% by weight of water, preferably at most 1% by weight of water,more preferably at most 0.5% by weight of water, and most preferably atmost 0.1% by weight of water.

A solvent or anti-solvent (as used in reactions, crystallization, etc.or lattice and/or adsorbed solvents) includes at least one of a C₁ to C₈alcohol, a C₂ to C₈ ether, a C₃ to C₇ ketone, a C₃ to C₇ ester, a C₁ toC₂ chlorocarbon, a C₂ to C₇ nitrile, a miscellaneous solvent, a C₅ toC₁₂ saturated hydrocarbon, and a C₆ to C₁₂ aromatic hydrocarbon.

The term C₁ to C₈ alcohol refers to a straight/branched and/orcyclic/acyclic alcohol having such number of carbons. The C₁ to C₈alcohol includes, but is not limited to, methanol, ethanol, n-propanol,isopropanol, isobutanol, hexanol, and cyclohexanol.

The term C₂ to C₈ ether refers to a straight/branched and/orcyclic/acyclic ether having such number of carbons. The C₂ to C₈ etherincludes, but is not limited to, dimethyl ether, diethyl ether,di-isopropyl ether, di-n-butyl ether, methyl-t-butyl ether (MTBE),tetrahydrofuran, and dioxane

The term C₃ to C₇ ketone refers to a straight/branched and/orcyclic/acyclic ketone having such number of carbons. The C₃ to C₇ ketoneincludes, but is not limited to, acetone, methyl ethyl ketone,propanone, butanone, methyl isobutyl ketone, methyl butyl ketone, andcyclohexanone.

The term C₃ to C₇ ester refers to a straight/branched and/orcyclic/acyclic ester having such number of carbons. The C₃ to C₇ esterincludes, but is not limited to, ethyl acetate, propyl acetate, n-butylacetate, etc.

The term C₁ to C₂ chlorocarbon refers to a chlorocarbon having suchnumber of carbons. The C₁ to C₂ chlorocarbon includes, but is notlimited to, chloroform, methylene chloride (DCM), carbon tetrachloride,1,2-dichloroethane, and tetrachloroethane.

A C₂ to C₇ nitrile refers to a nitrile have such number of carbons. TheC₂ to C₇ nitrile includes, but is not limited to, acetonitrile,propionitrile, etc.

A miscellaneous solvent refers to a solvent commonly employed in organicchemistry, which includes, but is not limited to, diethylene glycol,diglyme (diethylene glycol dimethyl ether), 1,2-dimethoxy-ethane,dimethylformamide, dimethylsulfoxide, ethylene glycol, glycerin,hexamethylphsphoramide, hexamethylphosphorous triame,N-methyl-2-pyrrolidinone, nitromethane, pyridine, triethyl amine, andacetic acid.

The term C₅ to C₁₂ saturated hydrocarbon refers to a straight/branchedand/or cyclic/acyclic hydrocarbon. The C₅ to C₁₂ saturated hydrocarbonincludes, but is not limited to, n-pentane, petroleum ether (ligroine),n-hexane, n-heptane, cyclohexane, and cycloheptane.

The term C₆ to C₁₂ aromatic refers to substituted and unsubstitutedhydrocarbons having a phenyl group as their backbone. Preferredhydrocarbons include benzene, xylene, toluene, chlorobenzene, o-xylene,m-xylene, p-xylene, xylenes, with toluene being more preferred.

The term “halo” or “halogen” as used herein, includes chloro, bromo,iodo and fluoro.

The term “blocking group” refers to a chemical group which exhibits thefollowing characteristics. The “group” is derived from a “protectingcompound.” Groups that are selective for primary hydroxyls oversecondary hydroxyls that can be put on under conditions consistent withthe stability of the phosphoramidate (pH 2-8) and impart on theresulting product substantially different physical properties allowingfor an easier separation of the 3′-phosphoramidate-5′-new group productfrom the unreacted desired compound. The group must react selectively ingood yield to give a protected substrate that is stable to the projectedreactions (see Protective Groups in Organic Synthesis, 3rd ed. T. W.Greene and P. G. M. Wuts, John Wiley & Sons, New York, N.Y., 1999).Examples of groups include, but are not limited to: benzoyl, acetyl,phenyl-substituted benzoyl, tetrahydropyranyl, trityl, DMT(4,4′-dimethoxytrityl), MMT (4-monomethoxytrityl), trimethoxytrityl,pixyl (9-phenylxanthen-9-yl), thiopixyl (9-phenylthioxanthen-9-yl) or9-(p-methoxyphenyl)xanthine-9-yl (MOX), etc.; C(O)-alkyl, C(O)Ph,C(O)aryl, CH₂O-alkyl, CH₂O-aryl, SO₂-alkyl, SO₂-aryl,tert-butyldimethylsilyl, tert-butyldiphenylsilyl. Acetals, such as MOMor THP and the like are exemplary groups. Fluorinated compounds are alsocontemplated in so far that they can be attached to the compound and canbe selectively removed by passing through a fluorous solid phaseextraction media (FluoroFlash™). A specific example includes afluorinated trityl analog,1-[4-(1H,1H,2H,2H-perfluorodecyl)phenyl)-1,1-diphenylmethanol. Otherfluorinated analogs of trityl, BOC, FMOC, CBz, etc. are alsocontemplated. Sulfonyl chlorides like p-toluenesulfonyl chloride canreact selectively on the 5′ position. Esters can be formed selectivelysuch as acetates and benzoates. Dicarboxylic anhydrides such as succinicanhydride and its derivatives can be used to generate an ester linkagewith a free carboxylic acid, such examples include, but are not limitedto, oxalyl, malonyl, succinyl, glutaryl, adipyl, pimelyl, superyl,azelayl, sebacyl, phthalyl, isophthalyl, terephthalyl, etc. The freecarboxylic acid increases the polarity dramatically and can also be usedas a handle to extract the reaction product into mildly basic aqueousphases such as sodium bicarbonate solutions. The phosphoramidate groupis relatively stable in acidic media, so groups requiring acidicreaction conditions, such as, tetrahydropyranyl, could also be used.

The term “protecting group” which is derived from a “protectingcompound,” has its plain and ordinary meaning, i.e., at least oneprotecting or blocking group is bound to at least one functional group(e.g., —OH, —NH₂, etc.) that allows chemical modification of at leastone other functional group. Examples of protecting groups, include, butare not limited to, benzoyl, acetyl, phenyl-substituted benzoyl,tetrahydropyranyl, trityl, DMT (4,4′-dimethoxytrityl), MMT(4-monomethoxytrityl), trimethoxytrityl, pixyl (9-phenylxanthen-9-yl)group, thiopixyl (9-phenylthioxanthen-9-yl) or9-(p-methoxyphenyl)xanthine-9-yl (MOX), etc.; C(O)-alkyl, C(O)Ph,C(O)aryl, C(O)O(lower alkyl), C(O)O(lower alkylene)aryl (e.g.,—C(O)OCH₂Ph), C(O)O-aryl, CH₂O-alkyl, CH₂O-aryl, SO₂-alkyl, SO₂-aryl,and a protecting group comprising at least one silicon atom, such as,tert-butyldimethylsilyl, tert-butyldiphenylsilyl, Si(loweralkyl)₂OSi(lower alkyl)₂OH (such as —Si(^(i)Pr)₂OSi(^(i)Pr)₂OH).

The term “protecting compound,” as used herein and unless otherwisedefined, refers to a compound that contains a “protecting group” andthat is capable of reacting with a compound that contains functionalgroups that are capable of being protected.

The term “leaving group”, as used herein, has the same meaning to theskilled artisan (Advanced Organic Chemistry: reactions, mechanisms andstructure—Fourth Edition by Jerry March, John Wiley and Sons Ed.; 1992pages 351-357) and represents a group which is part of and attached to asubstrate molecule; in a reaction where the substrate molecule undergoesa displacement reaction (with for example a nucleophile), the leavinggroup is then displaced. Examples of leaving groups include, but are notlimited to: halogen (F, Cl, Br, and I), preferably Cl, Br, or I;tosylate, mesylate, triflate, acetate, camphorsulfonate, aryloxide, andaryloxide substituted with at least one electron withdrawing group(e.g., p-nitrophenoxide, 2-chlorophenoxide, 4-chlorophenoxide,2,4-dinitrophenoxide, pentafluorophenoxide, etc.), etc. The term“electron withdrawing group” is accorded its plain meaning here.Examples of electron withdrawing groups include, but are not limited to,a halogen, —NO₂, —C(O)(lower alkyl), —C(O)(aryl), —C(O)O(lower alkyl),—C(O)O(aryl), etc.

The term “basic reagent”, as used herein, means a compound that iscapable of deprotonating a hydroxyl group. Examples of basic reagentsinclude, but are not limited to, a (lower alk)oxide ((lower alkyl)OM) incombination with an alcoholic solvent, where (lower alk)oxides include,but are not limited to, MeO⁻, EtO⁻, ^(n)PrO⁻, ^(i)PrO⁻, ^(t)BuO⁻,^(i)AmO-(iso-amyloxide), etc., and where M is an alkali metal cation,such as Li⁺, Na⁺, K⁺, etc. Alcoholic solvents include (lower alkyl)OH,such as, for example, MeOH, EtOH, ^(n)prOH, ^(i)PrOH, ^(t)BuOH,^(i)AmOH, etc. Non-alkoxy bases can also be used such as sodium hydride,sodium hexamethyldisilazane, lithium hexamethyldisilazane, lithiumdiisopropylamide, calcium hydride, sodium carbonate, potassiumcarbonate, cesium carbonate, DBU, DBN, and Grignard reagents, such as(lower alkyl)Mg(halogen), which include, but are not limited to, MeMgCl,MeMgBr, ^(t)BuMgCl, ^(t)BuMgBr, etc.

The term “base” embraces the term “basic reagent” and is meant to be acompound that is capable of deprotonating a proton-containing compound,i.e., a Bronsted base. In addition to the examples recited above,further examples of a base include, but are not limited to, pyridine,collidine, 2,6-(loweralkyl)-pyridine, dimethyl-aniline, imidazole,N-methyl-imidazole, pyrazole, N-methyl-pyrazole, triethylamine,di-isopropylethylamine, etc.

The term “electron-withdrawing group” is accorded its plain meaning.Examples of electron withdrawing groups include, but are not limited to,a halogen (F, Cl, Br, or I), —NO₂, —C(O)(lower alkyl), —C(O)(aryl),—C(O)O(lower alkyl), —C(O)O(aryl), etc.

The term “salts,” as described herein, refers to a compound comprising acation and an anion, which can produced by the protonation of aproton-accepting moiety and/or deprotonation of a proton-donatingmoiety. It should be noted that protonation of the proton-acceptingmoiety results in the formation of a cationic species in which thecharge is balanced by the presence of a physiological anion, whereasdeprotonation of the proton-donating moiety results in the formation ofan anionic species in which the charge is balanced by the presence of aphysiological cation.

The phrase “pharmaceutically acceptable salt” means a salt that ispharmaceutically acceptable. Examples of pharmaceutically acceptablesalts include, but are not limited to: (1) acid addition salts, formedwith inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, and the like; or formedwith organic acids such as glycolic acid, pyruvic acid, lactic acid,malonic acid, malic acid, maleic acid, fumaric acid, tartaric acid,citric acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonicacid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid, lauryl sulfuric acid,gluconic acid, glutamic acid, salicylic acid, muconic acid, and the likeor (2) basic addition salts formed with the conjugate bases of any ofthe inorganic acids listed above, wherein the conjugate bases comprise acationic component selected from among Na⁺, K⁺, Mg²⁺, Ca²⁺,NH_(g)R′″_(4-g) ⁺, in which R′″ is a C₁₋₃ alkyl and g is a numberselected from 0, 1, 2, 3, or 4. It should be understood that allreferences to pharmaceutically acceptable salts include solvent additionforms (solvates) or crystal forms (polymorphs) as defined herein, of thesame acid addition salt.

The term “alkyl” refers to an unbranched or branched chain, saturated,monovalent hydrocarbon residue containing 1 to 30 carbon atoms. The term“C₁-M alkyl” refers to an alkyl comprising 1 to M carbon atoms, where Mis an integer having one of the following values: 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30. In certain embodiments, the alkyl is a C₁₋₃₀ alkyl,such as C₁₋₂₂ alkyl, such as C₁₋₁₅ alkyl, such as C₁₋₉ alkyl, andfurther such as C₁₋₅ alkyl. The term “C₁₋₄ alkyl” refers to an alkylcontaining 1 to 4 carbon atoms. The term “lower alkyl” denotes astraight or branched chain hydrocarbon residue comprising 1 to 6 carbonatoms. “C₁₋₂₀ alkyl” as used herein refers to an alkyl comprising 1 to20 carbon atoms. “C₁₋₁₀ alkyl” as used herein refers to an alkylcomprising 1 to 10 carbons. Examples of alkyl groups include, but arenot limited to, lower alkyl groups include methyl, ethyl, propyl,i-propyl, n-butyl, i-butyl, t-butyl or pentyl, isopentyl, neopentyl,hexyl, heptyl, and octyl. The term (ar)alkyl or (heteroaryl)alkylindicate the alkyl group is optionally substituted by an aryl or aheteroaryl group respectively.

The term “C₁₋₁₀ haloalkyl” means a linear or branched, saturated,monovalent hydrocarbon group in which the term “alkyl” is as definedabove, and in which one or more of the hydrogen atoms are replaced,identically or differently, with a halogen atom. Preferably, saidhalogen atom is a fluorine atom. Said C₁₋₁₀ haloalkyl, particularly aC₁₋₃ haloalkyl group is, for example, fluoromethyl, difluoromethyl,trifluoromethyl, 2 fluoroethyl, 2,2 difluoroethyl, 2,2,2 trifluoroethyl,pentafluoroethyl, 3,3,3 trifluoropropyl or 1,3 difluoropropan 2 yl.

The term “alkenyl” refers to an unsubstituted hydrocarbon chain radicalhaving from 2 to 10 carbon atoms having one or two olefinic doublebonds, preferably one olefinic double bond. The term “C_(2-N) alkenyl”refers to an alkenyl comprising 2 to N carbon atoms, where N is aninteger having one of the following values: 3, 4, 5, 6, 7, 8, 9, or 10.The term “C₂₋₁₀ alkenyl” refers to an alkenyl comprising 2 to 10 carbonatoms. The term “C₂₋₄ alkenyl” refers to an alkenyl comprising 2 to 4carbon atoms. Examples include, but are not limited to, vinyl,1-propenyl, 2-propenyl (allyl) or 2-butenyl (crotyl). Furtherrepresentative alkenyl groups include, for example, an ethenyl-,prop-2-enyl-, (E)-prop-1-enyl-, (Z)-prop-1-enyl-, iso-propenyl-,but-3-enyl-, (E)-but-2-enyl-, (Z)-but-2-enyl-, (E)-but-1-enyl-,(Z)-but-1-enyl-, 2-methylprop-2-enyl-, 1-methylprop-2-enyl-,2-methylprop-1-enyl-, (E)-1-methylprop-1-enyl-,(Z)-1-methylprop-1-enyl-, buta-1,3-dienyl-, pent-4-enyl-,(E)-pent-3-enyl-, (Z)-pent-3-enyl-, (E)-pent-2-enyl-, (Z)-pent-2-enyl-,(E)-pent-1-enyl-, (Z)-pent-1-enyl-, 3-methylbut-3-enyl-,2-methylbut-3-enyl-, 1-methylbut-3-enyl-, 3-methylbut-2-enyl-,(E)-2-methylbut-2-enyl-, (Z)-2-methylbut-2-enyl-,(E)-1-methylbut-2-enyl-, (Z)-1-methylbut-2-enyl-,(E)-3-methylbut-1-enyl-, (Z)-3-methylbut-1-enyl-,(E)-2-methylbut-1-enyl-, (Z)-2-methylbut-1-enyl-,(E)-1-methylbut-1-enyl-, (Z)-1-methylbut-1-enyl-,1,1-dimethylprop-2-enyl-, 1-ethylprop-1-enyl-, 1-propylvinyl-,1-isopropylvinyl-, (E)-3,3-dimethylprop-1-enyl-,(Z)-3,3-dimethylprop-1-enyl-, penta-1,4-dienyl-, hex-5-enyl-,(E)-hex-4-enyl-, (Z)-hex-4-enyl-, (E)-hex-3-enyl-, (Z)-hex-3-enyl-,(E)-hex-2-enyl-, (Z)-hex-2-enyl-, (E)-hex-1-enyl-, (Z)-hex-1-enyl-,4-methylpent-4-enyl-, 3-methylpent-4-enyl-, 2-methylpent-4-enyl-,1-methylpent-4-enyl-, 4-methylpent-3-enyl-, (E)-3-methylpent-3-enyl-,(Z)-3-methylpent-3-enyl-, (E)-2-methylpent-3-enyl-,(Z)-2-methylpent-3-enyl-, (E)-1-methylpent-3-enyl-,(Z)-1-methylpent-3-enyl-, (E)-4-methylpent-2-enyl-,(Z)-4-methylpent-2-enyl-, (E)-3-methylpent-2-enyl-,(Z)-3-methylpent-2-enyl-, (E)-2-methylpent-2-enyl-,(Z)-2-methylpent-2-enyl-, (E)-1-methylpent-2-enyl-,(Z)-1-methylpent-2-enyl-, (E)-4-methylpent-1-enyl-,(Z)-4-methylpent-1-enyl-, (E)-3-methylpent-1-enyl-,(Z)-3-methylpent-1-enyl-, (E)-2-methylpent-1-enyl-,(Z)-2-methylpent-1-enyl-, (E)-1-methylpent-1-enyl-,(Z)-1-methylpent-1-enyl-, 3-ethylbut-3-enyl-, 2-ethylbut-3-enyl-,1-ethylbut-3-enyl-, (E)-3-ethylbut-2-enyl-, (Z)-3-ethylbut-2-enyl-,(E)-2-ethylbut-2-enyl-, (Z)-2-ethylbut-2-enyl-, (E)-1-ethylbut-2-enyl-,(Z)-1-ethylbut-2-enyl-, (E)-3-ethylbut-1-enyl-, (Z)-3-ethylbut-1-enyl-,2-ethylbut-1-enyl-, (E)-1-ethylbut-1-enyl-, (Z)-1-ethylbut-1-enyl-,2-propylprop-2-enyl-, 1-propylprop-2-enyl-, 2-isopropylprop-2-enyl-,1-isopropylprop-2-enyl-, (E)-2-propylprop-1-enyl-,(Z)-2-propylprop-1-enyl-, (E)-1-propylprop-1-enyl-,(Z)-1-propylprop-1-enyl-, (E)-2-isopropylprop-1-enyl-,(Z)-2-isopropylprop-1-enyl-, (E)-1-isopropylprop-1-enyl-,(Z)-1-isopropylprop-1-enyl-, hexa-1,5-dienyl- and1-(1,1-dimethylethyl-)ethenyl-group. Particularly, said group isethenyl- or prop-2-enyl-.

The term “C₂-C₆-alkynyl-” means a linear or branched, monovalenthydrocarbon group which contains one or more triple bonds, and whichcontains 2, 3, 4, 5 or 6 carbon atoms, preferably 2, 3 or 4 carbon atoms(“C₂-C₄-alkynyl-”) or 2 or 3 carbon atoms (“C₂-C₃-alkynyl-”).Representative C₂-C₆-alkynyl-groups include, for example, ethynyl-,prop-1-ynyl-, prop-2-ynyl-, but-1-ynyl-, but-2-ynyl-, but-3-ynyl-,pent-1-ynyl-, pent-2-ynyl, pent-3-ynyl-, pent-4-ynyl-, hex-1-ynyl-,hex-2-ynyl-, hex-3-ynyl-, hex-4-ynyl-, hex-5-ynyl-,1-methylprop-2-ynyl-, 2-methylbut-3-ynyl-, 1-methylbut-3-ynyl-,1-methylbut-2-ynyl-, 3-methylbut-1-ynyl-, 1-ethylprop-2-ynyl-,3-methylpent-4-ynyl-, 2-methylpent-4-ynyl-, 1-methylpent-4-ynyl-,2-methylpent-3-ynyl-, 1-methylpent-3-ynyl-, 4-methylpent-2-ynyl-,1-methylpent-2-ynyl-, 4-methylpent-1-ynyl-, 3-methylpent-1-ynyl-,2-ethylbut-3-ynyl-, 1-ethylbut-3-ynyl-, 1-ethylbut-2-ynyl-,1-propylprop-2-ynyl-, 1-isopropylprop-2-ynyl-, 2,2-dimethylbut-3-ynyl-,1,1-dimethylbut-3-ynyl-, 1,1-dimethylbut-2-ynyl- and3,3-dimethylbut-1-ynyl-group. Particularly, said alkynyl-group isethynyl-, prop-1-ynyl- or prop-2-ynyl-.

The term “lower alkoxy” means a linear or branched, saturated,monovalent group of formula (C₁-C₆-alkyl)-O—, in which the term“C₁-C₆-alkyl” is as defined above, e.g. a methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, sec-butoxy, isobutoxy, tert-butoxy, pentyloxy,isopentyloxy or n-hexyloxy group, or an isomer thereof.

The term “aryl,” as used herein, and unless otherwise specified, refersto substituted or unsubstituted phenyl (Ph), biphenyl, or naphthyl,preferably the term aryl refers to substituted or unsubstituted phenyl.The aryl group can be substituted with one or more moieties selectedfrom among hydroxyl, F, Cl, Br, I, amino, alkylamino, arylamino, alkoxy,aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid,phosphate, and phosphonate, either unprotected, or protected asnecessary, as known to those skilled in the art, for example, as taughtin T. W. Greene and P. G. M. Wuts, “Protective Groups in OrganicSynthesis,” 3rd ed., John Wiley & Sons, 1999.

The term “aryloxide,” as used herein, and unless otherwise specified,refers to substituted or unsubstituted phenoxide (PhO—),p-phenyl-phenoxide (p-Ph-PhO—), or naphthoxide, preferably the termaryloxide refers to substituted or unsubstituted phenoxide. Thearyloxide group can be substituted with one or more moieties selectedfrom among hydroxyl, F, Cl, Br, I, —C(O)(lower alkyl), —C(O)O(loweralkyl), amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano,sulfonic acid, sulfate, phosphonic acid, phosphate, and phosphonate,either unprotected, or protected as necessary, as known to those skilledin the art, for example, as taught in T. W. Greene and P. G. M. Wuts,“Protective Groups in Organic Synthesis,” 3rd ed., John Wiley & Sons,1999.

The term “C₃-C₁₀-cycloalkyl” means a saturated mono- or bicyclichydrocarbon ring which contains 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms(“C₃-C₁₀-cycloalkyl-”). Said C₃-C₁₀-cycloalkyl-group may be, forexample, a monocyclic hydrocarbon ring, e.g., a cyclopropyl-,cyclobutyl-, cyclopentyl-, cyclohexyl- or cycloheptyl-group, or abicyclic hydrocarbon ring, such as decalinyl-. Preferably, saidhydrocarbon ring is monocyclic and contains 3, 4, 5, 6 or 7 carbon atoms(“C₃-C₇-cycloalkyl-”), e.g., a cyclopropyl-, cyclobutyl-, cyclopentyl-,cyclohexyl- or cycloheptyl-group, or 3, 4, 5 or 6 carbon atoms(“C₃-C₆-cycloalkyl-”), e.g., a cyclopropyl-, cyclobutyl-, cyclopentyl-or cyclohexyl-group.

The term “heterocyclyl” or “heterocycloalkyl” means a saturated mono- orbicyclic hydrocarbon ring which contains 3, 4, 5, 6, 7, 8 or 9 carbonatoms, and which contains 1, 2, 3 or 4 heteroatoms which may beidentical or different, said heteroatoms preferably selected fromphosphorous, oxygen, nitrogen or sulfur, and wherein carbon atoms andheteroatoms add up to 4, 5, 6, 7, 8, 9 or 10 ring atoms in total, itbeing possible for said heterocycloalkyl-group to be attached to therest of the molecule via any one of the carbon atoms or, if present, anitrogen atom. “Heterospirocycloalkyl-”, “heterobicycloalkyl-” and“bridged heterocycloalkyl-”, as defined infra, are also included withinthe scope of this definition.

Preferably, a 4- to 10-membered heterocycloalkyl is monocyclic andcontains 3, 4, 5 or 6 carbon atoms, and one or two of theabove-mentioned heteroatoms, adding up to 4, 5, 6 or 7 ring atoms intotal (a “4- to 7-membered monocyclic heterocycloalkyl-”), or contains3, 4 or 5 carbon atoms, and one or two of the above-mentionedheteroatoms, adding up to 4, 5 or 6 ring atoms in total (a “4- to6-membered monocyclic heterocycloalkyl”), or contains 3, 4 or 5 carbonatoms, and one or two of the above-mentioned heteroatoms, adding up to 5or 6 ring atoms in total (a “5- to 6-membered monocyclicheterocycloalkyl”); it being possible for said heterocycloalkyl-group tobe attached to the rest of the molecule via any one of the carbon atomsor the nitrogen atoms, if present.

Exemplarily, without being limited thereto, said “monocyclicheterocycloalkyl”, can be a 4-membered ring, a “4-memberedheterocycloalkyl”, such as azetidinyl or oxetanyl; or a 5-membered ring,a “5-membered heterocycloalkyl”, such as tetrahydrofuranyl, dioxolinyl,pyrrolidinyl, imidazolidinyl, pyrazolidinyl- or pyrrolinyl-; or a6-membered ring, a “6-membered heterocycloalkyl”, such astetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinylor piperazinyl; or a 7-membered ring, a “7-membered heterocycloalkyl”,such as azepanyl, diazepanyl or oxazepanyl, for example.

The term “heteroaryl” means a monocyclic, bicyclic or tricyclic aromaticring system having 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ring atoms (a “5-to 14-membered heteroaryl” group), preferably 5, 6, 9 or 10 ring atoms,and which contains 1, 2, 3 or 4 heteroatoms, which may be identical ordifferent, said heteroatoms selected from oxygen, nitrogen and sulfur.Said heteroaryl group can be a 5-membered heteroaryl group, such as, forexample, thienyl-, furanyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl,pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl,thiadiazolyl or tetrazolyl; or a 6-membered heteroaryl group, such as,for example, pyridyl, pyridazinyl, pyrimidyl, pyrazinyl or triazinyl; ora benzo-fused 5-membered heteroaryl group, such as, for example,benzofuranyl, benzothienyl, benzoxazolyl, benzisoxazolyl,benzimidazolyl, benzothiazolyl, benzotriazolyl, indazolyl, indolyl orisoindolyl; or a benzo-fused 6-membered heteroaryl-group, such as, forexample, quinolinyl, quinazolinyl, isoquinolinyl, cinnolinyl,phthalazinyl or quinoxalinyl; or another bicyclic group, such as, forexample, indolizinyl, purinyl or pteridinyl; or a tricyclic heteroarylgroup, such as, for example, carbazolyl, acridinyl or phenazinyl.

Preferably, “heteroaryl-” is a monocyclic aromatic ring system having 5or 6 ring atoms and which contains at least one heteroatom, if more thanone, they may be identical or different, said heteroatom being selectedfrom oxygen, nitrogen and sulfur (“5- to 6-membered monocyclicheteroaryl-”), such as, for example, thienyl, furanyl, pyrrolyl,oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl,oxadiazolyl, triazolyl, thiadiazolyl, tetrazolyl, pyridyl, pyridazinyl,pyrimidyl, pyrazinyl or triazinyl.

In general, and unless otherwise mentioned, said heteroaryl groupsinclude all the possible isomeric forms thereof, e.g., the positionalisomers thereof. Thus, for some illustrative non-restricting example,the term pyridyl includes pyridin-2-yl, pyridin-3-yl and pyridin-4-yl;the term thienyl includes thien-2-yl and thien-3-yl. Furthermore, saidheteroaryl groups can be attached to the rest of the molecule via anyone of the carbon atoms, or, if applicable, a nitrogen atom, e.g.,pyrrol-1-yl, pyrazol-1-yl or imidazol-1-yl.

In general, and unless otherwise mentioned, the heteroaryl orheteroarylene groups include all possible isomeric forms thereof, e.g.,tautomers and positional isomers with respect to the point of linkage tothe rest of the molecule. Thus, for some illustrative non-restrictingexamples, the term pyridinyl includes pyridin-2-yl, pyridin-3-yl andpyridin-4-yl; or the term thienyl includes thien-2-yl and thien-3-yl.

The term “optionally substituted” means that the number of substituentscan be equal to or different from zero. Unless otherwise indicated, itis possible that optionally substituted groups are substituted with asmany optional substituents as can be accommodated by replacing ahydrogen atom with a non-hydrogen substituent on any available carbon ornitrogen atom. Commonly, it is possible for the number of optionalsubstituents, when present, to be 1, 2, 3, 4 or 5, in particular 1, 2 or3.

When groups in the compounds according to the invention are substituted,it is possible for said groups to be mono-substituted orpoly-substituted with substituent(s), unless otherwise specified. Withinthe scope of the present invention, the meanings of all groups whichoccur repeatedly are independent from one another. It is possible thatgroups in the compounds according to the invention are substituted withone, two, three, four or five identical or different substituents,particularly with one, two or three substituents.

The compounds of the present invention furthermore optionally containone or more asymmetric centrers, depending upon the location and natureof the various substituents desired. It is possible that one or moreasymmetric carbon atoms are present in the (R) or (S) configuration,which can result in racemic mixtures in the case of a single asymmetriccenter, and in diastereomeric mixtures in the case of multipleasymmetric centers.

Preferred compounds are those which produce the more desirablebiological activity. Separated, pure or partially purified isomers andstereoisomers or racemic or diastereomeric mixtures of the compounds ofthe present invention are also included within the scope of the presentinvention. The purification and the separation of such materials can beaccomplished by standard techniques known in the art.

If only one diastereomer displays the desired biological activity, and asecond disastereomer is inactive, the preferred isomer is the one whichproduces the more desirable biological activity. These separated, pureor partially purified isomers or racemic mixtures of the compounds ofthis invention are also included within the scope of the presentinvention. The purification and the separation of such materials can beaccomplished by standard techniques known in the art.

The optical isomers can be obtained by resolution of the racemicmixtures according to conventional processes, for example, by theformation of diastereoisomeric salts using an optically active acid orbase or formation of covalent diastereomers. Examples of appropriateacids are tartaric, diacetyltartaric, ditoluoyltartaric andcamphorsulfonic acid. Mixtures of diastereoisomers can be separated intotheir individual diastereomers on the basis of their physical and/orchemical differences by methods known in the art, for example, bychromatography or fractional crystallisation. The optically active basesor acids are then liberated from the separated diastereomeric salts. Adifferent process for separation of optical isomers involves the use ofchiral chromatography (e.g., HPLC columns using a chiral phase), with orwithout conventional derivatisation, optimally chosen to maximise theseparation of the enantiomers. Suitable HPLC columns using a chiralphase are commercially available, such as those manufactured by Daicel,e.g., Chiracel OD and Chiracel OJ, for example, among many others, whichare all routinely selectable. Enzymatic separations, with or withoutderivatisation, are also useful. The optically active compounds of thepresent invention can likewise be obtained by chiral syntheses utilizingoptically active starting materials, enantioselective catalyticreactions, and other suitable methods.

In order to distinguish different types of isomers from each otherreference is made to IUPAC Rules Section E (Pure Appl Chem 45, 11-30,1976).

The present invention includes all possible stereoisomers of thecompounds of the present invention as single stereoisomers, or as anymixture of said stereoisomers, in any ratio. Isolation of a singlestereoisomer, e.g., a single enantiomer or a single diastereomer, of acompound of the present invention may be achieved by any suitablemethod, such as chromatography, especially chiral chromatography, forexample.

Further, it is possible for the compounds of the present invention toexist as tautomers. For example, any compound of the present inventionwhich contains an pyrazol moiety as a heteroaryl group for example canexist as a 1H tautomer, or a 2H tautomer, or even a mixture in anyamount of the two tautomers, namely:

The present invention includes all possible tautomers of the compoundsof the present invention as single tautomers, or as any mixture of saidtautomers, in any ratio.

Further, the compounds of the present invention can exist as N-oxides,which are defined in that at least one nitrogen of the compounds of thepresent invention is oxidised. The present invention includes all suchpossible N-oxides.

The present invention also includes useful forms of the compounds of thepresent invention, such as metabolites, hydrates, solvates, prodrugs,salts, in particular pharmaceutically acceptable salts, and/orco-precipitates.

The compounds of the present invention can exist as a hydrate, or as asolvate, wherein the compounds of the present invention form a crystalthat contains molecules of polar solvents, in particular water, methanolor ethanol, for example, as structural element of the crystal lattice ofthe compounds. The molecules of polar solvents, in particular water, maybe present in a stoichiometric or non-stoichiometric ratio with themolecules of the compound. In the case of stoichiometric solvates, e.g.,a hydrate, hemi-, (semi-), mono-, sesqui-, di-, tri-, tetra-, penta-etc. solvates or hydrates, respectively, are possible. The presentinvention includes all such hydrates or solvates.

Further, it is possible for the compounds of the present invention toexist in free form, e.g., as a free base, or as a free acid, or as azwitterion, or to exist in the form of a salt. Said salt may be anysalt, either an organic or inorganic addition salt, particularly anypharmaceutically acceptable organic or inorganic addition salt, which iscustomarily used in pharmacy, or which is used, for example, forisolating or purifying the compounds of the present invention.

The term “subject” to which administration is contemplated includes, butis not limited to, humans (i.e., a male or female of any age group,e.g., a pediatric subject (e.g., infant, child, adolescent) or adultsubject (e.g., young adult, middle-aged adult or senior adult)) and/orother primates (e.g., cynomolgus monkeys, rhesus monkeys); mammals,including commercially relevant mammals such as cattle, pigs, horses,sheep, goats, cats, and/or dogs; and/or birds, including commerciallyrelevant birds such as chickens, ducks, geese, quail, and/or turkeys.

Moreover, the present invention also includes prodrugs of the compoundsaccording to the invention. The term “prodrugs” designates compoundswhich themselves can be biologically active or inactive, but areconverted (for example metabolically or hydrolytically) into compoundsaccording to the invention during their residence time in the body.Derivatives of the compounds disclosed herein, and the salts thereof,which are converted into a compound of formula (I), (II), or (III), or asalt thereof, in a biological system (bioprecursors or pro-drugs) arecovered by the invention. Said biological system may be, for example, amammalian organism, preferably a human subject. The bioprecursor is, forexample, converted into the compound of formula (I) or a salt thereof bymetabolic processes.

The term “prophylaxis” includes a use of the compound that, in astatistical sample, reduces the occurrence of the disorder or conditionin the treated sample relative to an untreated control sample, or delaysthe onset or reduces the severity of one or more symptoms of thedisorder or condition relative to the untreated control sample, whenadministered to prior to the onset of the disorder or condition.

The terms “treatment”, “treating”, “palliating” and “ameliorating” areused interchangeably herein. These terms refer to an approach forobtaining beneficial or desired results including, but not limited to,therapeutic benefit and/or a prophylactic benefit. By therapeuticbenefit is meant eradication or amelioration of the underlying disorderbeing treated. Also, a therapeutic benefit is achieved with theeradication or amelioration of one or more of the physiological symptomsassociated with the underlying disorder such that an improvement isobserved in the patient, notwithstanding that the patient can still beafflicted with the underlying disorder. For prophylactic benefit, thepharmaceutical compounds and/or compositions can be administered to apatient at risk of developing a particular disease, or to a patientreporting one or more of the physiological symptoms of a disease, eventhough a diagnosis of this disease may not have been made.

The term “preparation” or “dosage form” is intended to include bothsolid and liquid formulations of the active compound and one skilled inthe art will appreciate that an active ingredient can exist in differentpreparations depending on the desired dose and pharmacokineticparameters.

The term “excipient” as used herein refers to a compound that is used toprepare a pharmaceutical composition, and is generally safe, non-toxicand neither biologically nor otherwise undesirable, and includesexcipients that are acceptable for veterinary use as well as humanpharmaceutical use.

“Nicotinamide”, which corresponds to the following structure,

is one of the two principal forms of the B-complex vitamin niacin. Theother principal form of niacin is nicotinic acid; nicotinamide, ratherthan nicotinic acid, however, is the major substrate for nicotinamideadenine dinucleotide (NAD) biosynthesis in mammals, as discussed indetail herein. Nicotinamide, in addition to being known as niacinamide,is also known as 3-pyridinecarboxamide, pyridine-3-carboxamide,nicotinic acid amide, vitamin B3, and vitamin PP. Nicotinamide has amolecular formula of C₆H₆N₂O and its molecular weight is 122.13 Daltons.Nicotinamide is commercially available from a variety of sources.

“Nicotinamide Riboside” (NR), which corresponds to the followingstructure,

is characterized and a synthesized as described in, for instance, U.S.Pat. No. 8,106,184.

“Nicotinamide Mononucleotide” (NMN), which corresponds to the followingstructure,

is produced from nicotinamide in the NAD biosynthesis pathway, areaction that is catalyzed by Nampt. NMN is further converted to NAD inthe NAD biosynthesis pathway, a reaction that is catalyzed by Nmnat.Nicotinamide mononucleotide (NMN) has a molecular formula of C₁₁H₁₅N₂O₈Pand a molecular weight of 334.22. Nicotinamide mononucleotide (NMN) iscommercially available from such sources as Sigma-Aldrich (St. Louis,Mo.).

“Nicotinamide Adenine Dinucleotide” (NAD), which corresponds to thefollowing structure,

is produced from the conversion of nicotinamide to NMN, which iscatalyzed by Nampt, and the subsequent conversion of NMN to NAD, whichis catalyzed by Nmnat. Nicotinamide adenine dinucleotide (NAD) has amolecular formula of C₂₁H₂₇N₇O₁₄P₂ and a molecular weight of 663.43.Nicotinamide adenine dinucleotide (NAD) is commercially available fromsuch sources as Sigma-Aldrich (St. Louis, Mo.).Diseases, Disorders and Conditions

In certain embodiments, the invention relates to the use of compoundsand compositions comprising one or more compounds disclosed herein thatwork through the nicotinamide mononucleotide adenylyltransferase(Nmnat1) pathway or other pathways of NAD+ biosynthesis which havenutritional and/or therapeutic value in improving plasma lipid profiles,prevention of stroke, and/or prolonging life and well-being. Otherembodiments relate to a method for preventing or treating a disease orcondition associated with the nicotinamide mononucleotideadenylyltransferase (Nmnat1) pathway or other pathways of NAD+biosynthesis by administering a composition comprising one or morecompounds disclosed herein. Diseases or conditions which typically havealtered levels of NAD+ or its precursors which can be prevented ortreated by supplementing a diet or therapeutic treatment regime with acomposition comprising one or more compounds disclosed herein include,but are not limited to, lipid disorders, (e.g., dyslipidemia,hypercholesterolaemia or hyperlipidemia), stroke, type I and IIdiabetes, cardiovascular disease, and other physical problems associatedwith obesity.

Neurodegenerative Diseases

Axon degeneration occurs frequently in neurodegenerative diseases andperipheral neuropathies. The degeneration of transected axons is delayedin Wallerian degeneration slow (Wlds) mice with the overexpression of afusion protein with the nicotinamide adenine dinucleotide (NAD+)synthetic enzyme, nicotinamide mononucleotide adenylyltransferase(Nmnat1). Both Wld(s) and Nmnat1 themselves are functional in preventingaxon degeneration in neuronal cultures.

NAD+ levels decrease in injured, diseased, or degenerating neural cellsand preventing this NAD+ decline efficiently protects neural cells fromcell death. See, Araki & Milbrandt “Increased nuclear NAD+ biosynthesisand SIRT1 activation prevent axonal degeneration” Science. 2004 Aug. 13;305(5686):1010-3 and Wang et al., “A local mechanism mediatesNAD-dependent protection of axon degeneration” J Cell Biol.170(3):349-55 (2005) hereby incorporated by reference in their entirety.As the nicotinamide mononucleotide based compounds disclosed herein arecapable of increasing intracellular levels of NAD+, these compounds areuseful as a therapeutic or nutritional supplement in managing injuries,diseases, and disorders affecting the central nervous system and theperipheral nervous system, including, but not limited to, trauma orinjury to neural cells, diseases or conditions that harm neural cells,and neurodegenerative diseases or syndromes. The correlation ofincreased NAD+ synthesis with beneficial outcomes in neural injuries anddiseases or conditions has been discussed in, e.g., Stein et al.,“Expression of Nampt in Hippocampal and Cortical Excitatory Neurons IsCritical for Cognitive Function” The Journal of Neuroscience 201434(17):5800-5815; and Stein et al., “Specific ablation of Nampt in adultneural stem cells recapitulates their functional defects during aging”EMBO J. 2014 33:1321-1340.

Some neurodegenerative diseases, neurodegenerative syndromes, diseasesand conditions that harm neural cells, and injury to neural cells aredescribed below.

Essential tremor (ET) is the most common movement disorder. It is asyndrome characterized by a slowly progressive postural and/or kinetictremor, usually affecting both upper extremities.

Parkinson's disease (PD) is a progressive neurodegenerative disorderassociated with a loss of dopaminergic nigrostriatal neurons.

Alzheimer's disease (AD) is the most common form of dementia. It is aprogressive degenerative disease of the brain, strongly associated withadvanced age. Over time, people with the disease lose their ability tothink and reason clearly, judge situations, solve problems, concentrate,remember useful information, take care of themselves, and even speak. Anumber of neurodegenerative diseases such as Alzheimer's disease executetheir biological impact in the brain. It is preferred that nicotinamidemononucleotide based compounds disclosed herein are capable of passingthe blood-brain-barrier (BBB).

Huntington's disease (HD) is an incurable, adult-onset, autosomaldominant inherited disorder associated with cell loss within a specificsubset of neurons in the basal ganglia and cortex.

Ataxia is defined as an inability to maintain normal posture andsmoothness of movement. Neurologic symptoms and signs such as seizuresand movement disorders (e.g., dystonia, chorea) may accompany ataxia.

Catatonia is a state of apparent unresponsiveness to external stimuli ina person who is apparently awake.

Epilepsy is defined as a chronic condition characterized by spontaneous,recurrent seizures; seizure is defined as a clinical event associatedwith a transient, hypersynchronous neuronal discharge.

Neuroleptic malignant syndrome (NMS) refers to the combination ofhyperthermia, rigidity, and autonomic dysregulation that can occur as aserious complication of the use of antipsychotic drugs.

Chorea is an involuntary abnormal movement, characterized by abrupt,brief, nonrhythmic, nonrepetitive movement of any limb, often associatedwith nonpatterned facial grimaces. Chorea gravidarum (CG) is the termgiven to chorea occurring during pregnancy.

Cortical basal ganglionic degeneration (CBGD) clinical characteristicsinclude progressive dementia, parkinsonism, and limb apraxia.Dysfunction of the central or peripheral nervous system pathways maycause autonomic dysfunction.

Dystonia is a syndrome of sustained muscle contractions, usuallyproducing twisting and repetitive movements or abnormal postures.Writer's cramp is a form of task-specific focal dystonia.

Mental retardation (MR) is a condition in which intellectual capacity islimited significantly. Developmental disability describes a conditionthat limits an individual's ability to perform activities and roles asexpected in a certain social environment. Frequently, MR anddevelopmental disabilities are present simultaneously as a consequenceof brain damage.

Neuroacanthocytosis is a progressive neurologic disease characterized bymovement disorders, personality changes, cognitive deterioration, axonalneuropathy, and seizures. Most patients have acanthocytosis onperipheral blood smear at some point during the course of the disease.

Pelizaeus-Merzbacher disease (PMD) and X-linked spastic paraplegia type2 (SPG2) are at opposite ends of a clinical spectrum of X-linkeddiseases caused by mutations of the same gene, the proteolipid protein 1(PLP1) gene, and resulting in defective central nervous system (CNS)myelination. Clinical signs usually include some combination ofnystagmus, stridor, spastic quadriparesis, hypotonia, cognitiveimpairment, ataxia, tremor, and diffuse leukoencephalopathy on MRIscans.

Progressive supranuclear palsy (PSP), also known asSteele-Richardson-Olszewski syndrome, is a neurodegenerative diseasethat affects cognition, eye movements, and posture.

Striatonigral degeneration (SND) is a neurodegenerative disease thatrepresents a manifestation of multiple system atrophy (MSA). The othermanifestations are Shy-Drager syndrome (e.g., autonomic failurepredominates) and sporadic olivopontocerebellar degeneration (sOPCA,cerebellum predominates).

Ischemic stroke occurs due to a loss of blood supply to part of thebrain, initiating the ischemic cascade. Brain tissue ceases to functionif deprived of oxygen for more than 60 to 90 seconds and after a fewhours will suffer irreversible injury possibly leading to death of thetissue, i.e., infarction. Atherosclerosis may disrupt the blood supplyby narrowing the lumen of blood vessels leading to a reduction of bloodflow, by causing the formation of blood clots within the vessel, or byreleasing showers of small emboli through the disintegration ofatherosclerotic plaques. Embolic infarction occurs when emboli formedelsewhere in the circulatory system, typically in the heart as aconsequence of atria fibriliation, or in the carotid arteries. Thesebreak off, enter the cerebral circulation, then lodge in and occludebrain blood vessels.

Due to collateral circulation within the region of brain tissue affectedby ischemia, there is a spectrum of severity. Thus, part of the tissuemay immediately die while other parts may only be injured and couldpotentially recover. The ischemia area where tissue might recover isreferred to as the ischemic penumbra.

As oxygen or glucose becomes depleted in ischemic brain tissue, theproduction of high energy phosphate compounds such as adeninetriphosphate (ATP) fails, leading to failure of energy dependentprocesses necessary for tissue cell survival. This sets off a series ofinterrelated events that result in cellular injury and death. Theseinclude the failure of mitochondria, which can lead further towardenergy depletion and may trigger cell death due to apoptosis. Otherprocesses include the loss of membrane ion pump function leading toelectrolyte imbalances in brain cells. There is also the release ofexcitatory neurotransmitters, which have toxic effects in excessiveconcentrations.

Spinal cord injury, or myelopathy, is a disturbance of the spinal cordthat results in loss of sensation and mobility. The two common types ofspinal cord injury are: trauma: automobile accidents, falls, gunshots,diving accidents, etc. and disease: polio, spina bifida, tumors,Friedreich's ataxia, etc. It is important to note that the spinal corddoes not have to be completely severed for there to be a loss offunction. In fact, the spinal cord remains intact in most cases ofspinal cord injury.

Traumatic brain injury (TBI), also called intracranial injury, or simplyhead injury, occurs when a sudden trauma causes brain damage. TBI canresult from a closed head injury or a penetrating head injury and is oneof two subsets of acquired brain injury (ABI). The other subset isnon-traumatic brain injury (e.g., stroke, meningitis, anoxia). Parts ofthe brain that can be damaged include the cerebral hemispheres,cerebellum, and brain stem. Symptoms of a TBI can be mild, moderate, orsevere, depending on the extent of the damage to the brain. Outcome canbe anything from complete recovery to permanent disability or death. Acoma can also affect a child's brain. The damage from TBI can be focal,confined to one area of the brain, or diffuse, involving more than onearea of the brain. Diffuse trauma to the brain is frequently associatedwith concussion (a shaking of the brain in response to sudden motion ofthe head), diffuse axonal injury, or coma. Localized injuries may beassociated with neurobehavioral manifestations, hemiparesis or otherfocal neurologic deficits.

Another insult to the brain that can cause injury is anoxia. Anoxia is acondition in which there is an absence of oxygen supply to an organ'stissues, even if there is adequate blood flow to the tissue. Hypoxiarefers to a decrease in oxygen supply rather than a complete absence ofoxygen, and ischemia is inadequate blood supply, as is seen in cases inwhich the brain swells. In any of these cases, without adequate oxygen,a biochemical cascade called the ischemic cascade is unleashed, and thecells of the brain can die within several minutes. This type of injuryis often seen in near-drowning victims, in heart attack patients(particularly those who have suffered a cardiac arrest), or in peoplewho suffer significant blood loss from other injuries that then causes adecrease in blood flow to the brain due to circulatory (hypovolemic)shock.

Regulating Blood Glucose Concentration

Provided herein is a process for regulating the concentration of bloodglucose in a mammal. As utilized herein, regulating the concentration ofblood glucose refers to any increase, decrease, and/or maintenance in orof the concentration of blood glucose as compared to a previouslydetermined level.

Compounds of the present invention may be administered to a mammal inneed of such treatment. For example, the mammal may require an increasein blood glucose concentration. Alternatively, the mammal may require adecrease in blood glucose concentration. Or, the mammal may requiremaintenance of blood glucose concentration above, at, or below aparticular level or within a particular range (e.g., through a series ofincreases and/or decreases, or through no increases or decreases). Theblood glucose concentration-regulating compounds may also beadministered to a mammal as a prophylactic measure; that is, the mammalis in need of treatment to prevent or delay the occurrence or onset of amedical condition such as, for example, type 1 or type 2 diabetes.

The ability to regulate the concentration of blood glucose in a mammalaccording to the processes described herein (e.g., by administering to amammal a blood glucose regulating amount of a compound of the presentinvention may be advantageous in the treatment and/or prevention of avariety of complications, diseases, and/or illnesses. The role ofincreased NAD+ levels on metabolic diseases and conditions has beendescribed in, for example, Yoshino et al., “Nicotinamide mononucleotide,a key NAD+ intermediate, treats the pathophysiology of diet- andage-induced diabetes” Cell Metab. 2011 14:528-536; and Garten, et al.,“Nampt: Linking NAD biology, metabolism, and cancer” Trends EndocrinolMetab. 2009 20(3):130-138. In general, the present invention may beutilized to treat a variety of acute, intermediate stage, and chronicconditions that may be affected by systemic NAD biosynthesis eitherdirectly or indirectly.

For example, the regulation of blood glucose concentration may beeffective in the treatment and/or prophylaxis of such medical conditionsas brain ischemia-induced hypoglycemia, hypoglycemic brain injury causedby, e.g., congenital hyperinsulinism in children, and/or otherconditions that severely reduce blood glucose levels. Alternatively, theregulation of blood glucose concentration may be effective incounteracting the effects of the injection of an excessive amount ofinsulin, or an insufficient dietary or vitamin intake (e.g.,deficiencies in vitamin B3 (niacin, which is derived from nicotinic acidand nicotinamide) can result in pellagra, the classic niacin deficiencydisease, characterized by bilateral dermatitis, diarrhea, and dementia).

Further, regulation of blood glucose concentration may be effective inthe treatment and/or prophylaxis of hypoglycemia, hyperglycemia,impaired glucose tolerance, impaired fasting glucose, and type 1 andtype 2 diabetes.

The regulation of blood glucose concentration according to the methodsdescribed herein may also be advantageous in counteracting the effectsof blood glucose concentration-decreasing drugs such as acetaminophen,alcohol, anabolic steroids, clofibrate, disopyramide, gemfibrozil,monoamine oxidase inhibitors (MAOIs), pentamidine, or sulfonylureamedications (such as glipizide, glyburide, and glimepiride).

Other conditions having a plausible connection to NAD biosynthesis, suchas dementia, may also be beneficially treated and/or prevented by bloodglucose regulation. See, e.g., Guest, et al., “Changes in OxidativeDamage, Inflammation and [NAD(H)] with Age in Cerebrospinal Fluid” PLOSOne. January 2014 9(1): e85335.

The increase, decrease, and/or maintenance of blood glucoseconcentration can be quantified, for example, by percentage above,below, or in between one or more previously determined levels, or can bequantified by a particular blood glucose concentration or a rangethereof.

For example, the blood glucose concentration may be increased to atleast about 5% above a previously determined level; to at least about10% above a previously determined level; to at least about 25% above apreviously determined level; to at least about 50% above a previouslydetermined level; to at least about 75% above a previously determinedlevel; to at least about 100% above a previously determined level; to atleast about 150% above a previously determined level; or to at leastabout 200% above a previously determined level. By way of anotherexample, the blood glucose concentration may be decreased to at leastabout 5% below a previously determined level; to at least about 10%below a previously determined level; to at least about 25% below apreviously determined level; to at least about 50% below a previouslydetermined level; to at least about 75% below a previously determinedlevel; to at least about 100% below a previously determined level; to atleast about 150% below a previously determined level; or to at leastabout 200% below a previously determined level. By way of yet anotherexample, the blood glucose concentration may be maintained (e.g., by aseries of increases and/or decreases, or by no increases and/ordecreases) at a concentration that is no more than about 50% greater orabout 50% less than a previously determined level; e.g., no more thanabout 40% greater or about 40% less; no more than about 30% greater orabout 30% less; no more than about 20% greater or about 20% less; nomore than about 10% greater or about 10% less; or no more than about 5%greater or about 5% less.

Alternatively, the blood glucose concentration may be maintained (e.g.,by a series of increases and/or decreases, or by no increases and/ordecreases) at, above, or below a particular blood glucose concentrationor within a desired range of blood glucose concentrations. For example,the blood glucose concentration may be maintained at a concentration ofgreater than about 60 mg/dL; greater than about 70 mg/dL; greater thanabout 100 mg/dL; greater than about 110 mg/dL; or greater than about 125mg/dL. Alternatively, the blood glucose concentration may be maintainedat a concentration of less than about 200 mg/dL; less than about 175mg/dL; less than about 150 mg/dL; less than about 125 mg/dL; less thanabout 110 mg/dL; or less than about 100 mg/dL. By way of anotherexample, the blood glucose concentration may be maintained at aconcentration of from about 60 mg/dL to about 140 mg/dL; from about 90mg/dL to about 130 mg/dL; from about 100 mg/dL to about 125 mg/dL; orfrom about 110 mg/dL to about 125 mg/dL.

Drug Toxicity

In some embodiments, the invention relates to the use of a nicotinamidemononucleotide based derivative to prevent adverse effects and protectcells from toxicity.

Toxicity may be an adverse effect of radiation or external chemicals onthe cells of the body. Examples of toxins are pharmaceuticals, drugs ofabuse, and radiation, such as UV or X-ray light. Both radiative andchemical toxins have the potential to damage biological molecules suchas DNA. This damage typically occurs by chemical reaction of theexogenous agent or its metabolites with biological molecules, orindirectly through stimulated production of reactive oxygen species(e.g., superoxide, peroxides, hydroxyl radicals). Repair systems in thecell excise and repair damage caused by toxins.

Enzymes that use NAD+ play a part in the DNA repair process.Specifically, the poly(ADP-ribose) polymerases (PARPs), particularlyPARP-1, are activated by DNA strand breaks and affect DNA repair. ThePARPs consume NAD+ as an adenosine diphosphate ribose (ADPR) donor andsynthesize poly(ADP-ribose) onto nuclear proteins such as histones andPARP itself. Although PARP activities facilitate DNA repair,overactivation of PARP can cause significant depletion of cellular NAD+,leading to cellular necrosis. The apparent sensitivity of NAD+metabolism to genotoxicity has led to pharmacological investigationsinto the inhibition of PARP as a means to improve cell survival.Numerous reports have shown that PARP inhibition increases NAD+concentrations in cells subject to genotoxicity, with a resultingdecrease in cellular necrosis. See, e.g., Fang, et al., DefectiveMitophagy in XPA via PARP-1 Hyperactivation and NAD+/SIRT1 Reduction.Cell 2014 157:882-896. Nevertheless, cell death from toxicity stilloccurs, presumably because cells are able to complete apoptotic pathwaysthat are activated by genotoxicity. Thus, significant cell death isstill a consequence of DNA/macromolecule damage, even with inhibition ofPARP. This consequence suggests that improvement of NAD+ metabolism ingenotoxicity can be partially effective in improving cell survival butthat other proteins that modulate apoptotic sensitivity, such assirtuins, may also play important roles in cell responses to genotoxins.

Physiological and biochemical mechanisms that determine the effects ofchemical and radiation toxicity in tissues are complex, and evidenceindicates that NAD+ metabolism is an important aspect of cell stressresponse pathways. For example, upregulation of NAD+ metabolism, vianicotinamide/nicotinic acid mononucleotide (NMNAT) overexpression, hasbeen shown to protect against neuron axonal degeneration, andnicotinamide used pharmacologically has been recently shown to provideneuron protection in a model of fetal alcohol syndrome and fetalischemia. Such protective effects could be attributable to upregulatedNAD+ biosynthesis, which increases the available NAD+ pool subject todepletion during genotoxic stress. This depletion of NAD+ is mediated byPARP enzymes, which are activated by DNA damage and can deplete cellularNAD+, leading to necrotic death. Another mechanism of enhanced cellprotection that could act in concert with upregulated NAD+ biosynthesisis the activation of cell protection transcriptional programs regulatedby sirtuin enzymes.

Aging/Stress

In certain embodiments, the invention provides a method extending thelifespan of a cell, extending the proliferative capacity of a cell,slowing aging of a cell, promoting the survival of a cell, delayingcellular senescence in a cell, mimicking the effects of calorierestriction, increasing the resistance of a cell to stress, orpreventing apoptosis of a cell, by contacting the cell with anicotinamide mononucleotide based derivative compound. Recent studieshave demonstrated the role NAD+ plays in the aging process and inage-related diseases and conditions. See, e.g., Imai, et al., “NAD+ andsirtuins in aging and disease” Trends in Cell Biol. 2014 24(8): 464-471;and Gomes, et al. “Declining NAD+ Induces a Pseudohypoxic StateDisrupting Nuclear-Mitochondrial Communication during Aging” Cell 2013155:1624-1638.

The methods described herein may be used to increase the amount of timethat cells, particularly primary cells (e.g., cells obtained from anorganism, e.g., a human), may be kept alive in an ex vivo cell culture.Embryonic stem (ES) cells and pluripotent cells, and cellsdifferentiated therefrom, may also be treated with a nicotinamidemononucleotide based or derivative compound to keep the cells, orprogeny thereof, in culture for longer periods of time. Such cells canalso be used for transplantation into a subject, e.g., after ex vivomodification.

In certain embodiments, cells that are intended to be preserved for longperiods of time may be treated with a nicotinamide mononucleotide basedderivative compound. The cells may be in suspension (e.g., blood cells,serum, biological growth media, etc.) or in tissues or organs in asubject. For example, blood collected from an individual for purposes oftransfusion may be treated with a nicotinamide mononucleotide basedderivative compound to preserve the blood cells for longer periods oftime. Additionally, blood to be used for forensic purposes may also bepreserved using a nicotinamide mononucleotide based derivative compound.Other cells that may be treated to extend their lifespan or protectagainst apoptosis include cells for consumption, e.g., cells fromnon-human mammals (such as meat) or plant cells (such as vegetables).

Nicotinamide mononucleotide based derivative compounds may also beapplied during developmental and growth phases in mammals, plants,insects or microorganisms, in order to, e.g., alter, retard oraccelerate the developmental and/or growth process.

In certain embodiments, nicotinamide mononucleotide based derivativecompounds may be used to treat cells useful for transplantation or celltherapy, including, for example, solid tissue grafts, organ transplants,cell suspensions, stem cells, bone marrow cells, etc. The cells ortissue may be an autograft, an allograft, a syngraft or a xenograft. Thecells or tissue may be treated with the nicotinamide mononucleotidebased derivative compound prior to administration/implantation,concurrently with administration/implantation, and/or postadministration/implantation into a subject. The cells or tissue may betreated prior to removal of the cells from the donor individual, ex vivoafter removal of the cells or tissue from the donor individual, or postimplantation into the recipient. For example, the donor or recipientindividual may be treated systemically with a nicotinamidemononucleotide based derivative compound or may have a subset ofcells/tissue treated locally with a nicotinamide mononucleotide basedderivative compound. In certain embodiments, the cells or tissue (ordonor/recipient individuals) may additionally be treated with anothertherapeutic agent useful for prolonging graft survival, such as, forexample, an immunosuppressive agent, a cytokine, an angiogenic factor,etc.

In certain embodiments, cells may be treated with a nicotinamidemononucleotide based derivative compound that increases the level ofNAD+ in vivo, e.g., to increase their lifespan or prevent apoptosis. Forexample, skin can be protected from aging (e.g., developing wrinkles,loss of elasticity, etc.) by treating skin or epithelial cells with anicotinamide mononucleotide based derivative compound that increases thelevel of intracellular NAD+. In exemplary embodiments, skin is contactedwith a pharmaceutical or cosmetic composition comprising a nicotinamidemononucleotide based derivative compound that increases the level ofintracellular NAD+. Exemplary skin afflictions or skin conditions thatmay be treated in accordance with the methods described herein includedisorders or diseases associated with or caused by inflammation, sundamage or natural aging. For example, the compositions find utility inthe prevention or treatment of contact dermatitis (including irritantcontact dermatitis and allergic contact dermatitis), atopic dermatitis(also known as allergic eczema), actinic keratosis, keratinizationdisorders (including eczema), epidermolysis bullosa diseases (includingpenfigus), exfoliative dermatitis, seborrheic dermatitis, erythemas(including erythema multiforme and erythema nodosum), damage caused bythe sun or other light sources, discoid lupus erythematosus,dermatomyositis, psoriasis, skin cancer and the effects of naturalaging. In other embodiments, a nicotinamide mononucleotide basedderivative compound that increases the level of intracellular NAD+ maybe used for the treatment of wounds and/or burns to promote healing,including, for example, first-, second- or third-degree burns and/orthermal, chemical or electrical burns. The formulations may beadministered topically, to the skin or mucosal tissue, as an ointment,lotion, cream, microemulsion, gel, solution or the like, as furtherdescribed herein, within the context of a dosing regimen effective tobring about the desired result.

Topical formulations comprising one or more nicotinamide mononucleotidebased derivative compounds that increase the level of intracellular NAD+may also be used as preventive, e.g., chemopreventive, compositions.When used in a chemopreventive method, susceptible skin is treated priorto any visible condition in a particular individual.

In certain embodiments, a nicotinamide mononucleotide based derivativecompound that increases the level of intracellular NAD+ may be used fortreating or preventing a disease or condition induced or exacerbated bycellular senescence in a subject; methods for decreasing the rate ofsenescence of a subject, e.g., after onset of senescence; methods forextending the lifespan of a subject; methods for treating or preventinga disease or condition relating to lifespan; methods for treating orpreventing a disease or condition relating to the proliferative capacityof cells; and methods for treating or preventing a disease or conditionresulting from cell damage or death. In certain embodiments, the methoddoes not act by decreasing the rate of occurrence of diseases thatshorten the lifespan of a subject. In certain embodiments, a method doesnot act by reducing the lethality caused by a disease, such as cancer.

In certain embodiments, a nicotinamide mononucleotide based derivativecompound that increases the level of intracellular NAD+ may beadministered to a subject in order to generally increase the lifespan ofits cells and to protect its cells against stress and/or againstapoptosis. Treating a subject with a compound described herein may besimilar to subjecting the subject to hormesis, i.e., mild stress that isbeneficial to organisms and may extend their lifespan.

A nicotinamide mononucleotide based derivative compound that increasesthe level of intracellular NAD+ can also be administered to subjects fortreatment of diseases, e.g., chronic diseases, associated with celldeath, in order to protect the cells from cell death. Exemplary diseasesinclude those associated with neural cell death, neuronal dysfunction,or muscular cell death or dysfunction, such as Parkinson's disease,Alzheimer's disease, multiple sclerosis, amyotropic lateral sclerosis,and muscular dystrophy; AIDS; fulminant hepatitis; diseases linked todegeneration of the brain, such as Creutzfeld-Jakob disease, retinitispigmentosa and cerebellar degeneration; myelodysplasis such as aplasticanemia; ischemic diseases such as myocardial infarction and stroke;hepatic diseases such as alcoholic hepatitis, hepatitis B and hepatitisC; joint-diseases such as osteoarthritis; atherosclerosis; alopecia;damage to the skin due to UV light; lichen planus; atrophy of the skin;cataract; and graft rejections. Cell death can also be caused bysurgery, drug therapy, chemical exposure or radiation exposure.

A nicotinamide mononucleotide based derivative compound that increasesthe level of intracellular NAD+ can also be administered to a subjectsuffering from an acute disease, e.g., damage to an organ or tissue,e.g., a subject suffering from stroke or myocardial infarction or asubject suffering from a spinal cord injury. A nicotinamidemononucleotide based derivative compound that increases the level ofintracellular NAD+ may also be used to repair an alcoholic's liver.

Cardiovascular Disease

In certain embodiments, the invention provides methods for treatingand/or preventing a cardiovascular disease by administering to a subjectin need thereof a nicotinamide mononucleotide based derivative compoundthat increases the level of intracellular NAD+. The benefits of NAD+ intreating cardivasular diseases has been described in several studies,such as Borradaile, et al., “NAD+, Sirtuins, and Cardiovascular Disease”Current Pharmaceutical Design 2016 15(1):110-117.

Cardiovascular diseases that can be treated or prevented by anicotinamide mononucleotide based derivative compound that increases thelevel of intracellular NAD+ include cardiomyopathy or myocarditis; suchas idiopathic cardiomyopathy, metabolic cardiomyopathy, alcoholiccardiomyopathy, drug-induced cardiomyopathy, ischemic cardiomyopathy,and hypertensive cardiomyopathy. Also treatable or preventable usingcompounds and methods described herein are atheromatous disorders of themajor blood vessels (macrovascular disease) such as the aorta, thecoronary arteries, the carotid arteries, the cerebrovascular arteries,the renal arteries, the iliac arteries, the femoral arteries, and thepopliteal arteries. Other vascular diseases that can be treated orprevented include those related to platelet aggregation, the retinalarterioles, the glomerular arterioles, the vasa nervorum, cardiacarterioles, and associated capillary beds of the eye, the kidney, theheart, and the central and peripheral nervous systems.

Yet other disorders that may be treated with a nicotinamidemononucleotide based derivative compound that increases the level ofintracellular NAD+ include restenosis, e.g., following coronaryintervention, and disorders relating to an abnormal level of highdensity and low density cholesterol.

Circadian Rhythm

The circadian clock is encoded by a transcription-translation feedbackloop that synchronizes behavior and metabolism with the light-darkcycle. It has been unexpectedly discovered that both the rate-limitingenzyme in mammalian NAD+ biosynthesis, nicotinamidephosphoribosyltransferase (NAMPT), and levels of NAD+, display circadianoscillations which are regulated by the core clock machinery in mice.Inhibition of NAMPT promotes oscillation of the clock gene Per2 byreleasing CLOCK:BMAL1 from suppression by SIRT1. In turn, the circadiantranscription factor CLOCK binds to and up-regulates Nampt, thuscompleting a feedback loop involving NAMPT/NAD⁺ and SIRT1/CLOCK:BMAL1.See, e.g., Ramsey et al., “Circadian clock feedback cycle throughNAMPT-mediated NAD+ biosynthesis” Science 2009 324:651-654.

Thus, the periodic variation in NAMPT-mediated NAD+ biosynthesissuggests that it impacts physiologic cycles and possibly the sleep-wakeand fasting-feeding cycle. Without being bound by a single theory, it isbelieved that NAD+ serves as a critical “metabolic oscillator” for therhythmic regulation of response to environmental cues through control ofSIRT1 activity. Compounds disclosed herein may be used to affect acircadian feedback loop through NAMPT-mediated NAD+ biosynthesis and/ora pathway underlying the temporal coupling of metabolic, physiologic,and circadian cycles in mammals.

The recognition of a regulatory pathway involvingNAMPT/NAD+-SIRT1/CLOCK:BMAL1 has broad implications for understandinghow physiologic and behavioral cycles are coordinated with theenvironmental light-dark cycle. For instance, during sleep, when animalsare normally quiescent and fasting, the levels of NAMPT steadilyincrease, peaking at the beginning of the wakefulness period andcoinciding with feeding. As a result of the increase in NAMPT, NAD+rises to stimulate SIRT1, which orchestrates an appropriate metabolicresponse in liver involving a switch from catabolic to anabolicpathways.

In certain embodiments, the present invention provides methods forregulation of the core clock machinery (sometimes also referred to asthe circadian clock) of a mammal, thereby affecting behaviors,activities, and/or biological functions that occur in or are affected bya diurnal or circadian cycle and that are regulated, at least in part,by the circadian clock. Generally, the methods involve theadministration of a therapeutic or prophylactic amount of a circadianclock-regulating compound to a patient or mammal in need of regulationof the circadian clock.

The methods of treatment disclosed herein are generally directed tomethods of regulating the circadian clock, thereby regulating oraffecting biological functions that are regulated by (sometimes alsosaid to be affected by, affiliated with, or mediated by) the activity ofthe circadian clock. Typically, these biological functions display apattern of activity and inactivity that is generally repeatedapproximately every 24 hours, oscillating between “active” and“inactive” states during the 24 hour period.

Thus, the present invention provides methods of regulating the activityof the circadian clock by administering to a mammal in need thereof acircadian-clock regulating compound. Generally, the regulation of theactivity of the circadian clock is the result of the regulation ofCLOCK:BMAL1, which is achieved according to the present methods byregulating the activity of SIRT1. The activity of SIRT1 is generallyregulated according to the present methods by administration of acircadian clock-regulating compound, and in certain embodiments, byadministration of a compound that affects the NAD+ pathway. Theregulation of the circadian clock thereby permits regulation ofactivities mediated by the circadian clock.

According to the present invention, the activity of the circadian clockmay be increased, decreased, or maintained by the administration of acircadian clock-regulating compound. Accordingly, biological functions(sometimes also referred to as biological activities) that are regulatedby the activity of the circadian clock may also be increased, decreased,or maintained. In addition, these biological functions may also be timeshifted; that is to say, an activity that typically occurs during aparticular period, such as for example, during daytime or daylight hours(sometimes also referred to as the light cycle) or during the night ornighttime hours (sometimes also referred to as the dark cycle) may beshifted such that the activity occurs during the dark or light cycle,respectively, instead.

Any of a number of biological functions that are typically affected bythe activity of the circadian clock may be regulated by the methods ofthe present invention. Thus, the present methods may be used to treatdisorders or disease states that are the result of, for example, theirregular, inadequate, or pathological function of the circadian clock.Similarly, the present methods may be used to treat disorders orsymptomatology caused by exogenous factors that affect the properfunction or activity of the circadian clock or that require a“resetting” of the clock. For example, administration of circadianclock-regulating compound to a patient experiencing a metabolic disorderprovides therapeutic benefit not only when the patient's serum NMN orNAD level is increased, but also when an improvement is observed in thepatient with respect to other disorders that accompany the metabolicdisorder, like weight loss or gain. In some treatment regimens, thecircadian clock-regulating compound of the invention may be administeredto a patient at risk of developing a disorder as described herein or toa patient reporting one or more of the physiological symptoms of such adisorder, even though a diagnosis of a metabolic disorder may not havebeen made.

Examples of disorders, disease states, or symptomatology that may betreated according to the methods of the present invention include, butare not limited to, travel to or across one or more time zones, a changein work shifts, night shift work, or a change in the physical status ofa mammal caused by, for example, pregnancy or administration ofmedications of any kind. Accordingly, the methods of the presentinvention may be used to treat or prevent disorders, symptoms ofdisorders, or symptoms caused by exogenous factors. Such disorders andsymptoms may include, for example, metabolic disorders, such as impropercycling or timing of feeding and fasting cycles, hyperglycemia,hypoglycemia, or diabetes; sleep disorders, such as insomnia, advancedsleep phase syndrome, delayed sleep phase syndrome, inconsistentsleep/wake cycles, or narcolepsy or to improve wakefulness inindividuals suffering from excessive sleepiness; and symptoms caused byexogenous factors, such as, travel to or across one or more time zones(jet lag), shifting into or out of daylight savings time, a change inwork shifts or night shift work, pregnancy, or medications being takenfor unrelated diseases or disorders.

Accordingly, in certain embodiments, the present invention is directedto a method of regulating a biological function in a mammal, thefunction being affected by the circadian clock. The method comprisesadministering a therapeutic or prophylactic (sometimes also referred toas a circadian clock-regulating) amount of a circadian clock-regulatingcompound to the mammal. The biological function can be, for example, anyone of the biological functions described herein. In certainembodiments, the invention comprises a method of treating a metabolicdisorder in a mammal and comprises administering a therapeutic orprophylactic amount of a circadian clock-regulating compound to themammal. In other embodiments, the invention comprises a method oftreating a disorder in a mammal mediated by the function of thecircadian clock and comprises administering a therapeutic orprophylactic amount of a circadian clock-regulating compound to themammal. According to any one of these embodiments, the circadianclock-regulating compound may be, for example, nicotinamide,nicotinamide mononucleotide (NMN), nicotinamide adenine dinucleotide(NAD); salts and prodrugs thereof; nicotinamidephosphoribosyltransferase (NAMPT); and combinations thereof, asdescribed in greater detail below. In other embodiments, the circadianclock-regulating compound may be an antagonist of any one of thecompounds listed above, thereby exacting an effect opposite that ofnicotinamide, nicotinamide mononucleotide (NMN), nicotinamide adeninedinucleotide (NAD); salts and prodrugs thereof; nicotinamidephosphoribosyltransferase (NAMPT); and combinations thereof.

In certain embodiments, the present invention is directed to a method ofregulating metabolic activity of a mammal comprising administering tothe mammal a therapeutic amount of a circadian clock-regulatingcompound. In certain embodiments, the metabolic activity of the mammalis increased. In other embodiments, the metabolic activity is decreased.In yet other embodiments, the metabolic activity of the mammal ismaintained at a desired level, thereby preventing fluctuations inactivity/inactivity. In still other embodiments, the metabolic activityis caused to occur in the light cycle (as opposed to its typicaloccurrence in the dark cycle). In other embodiments, the metabolicactivity is caused to occur in the dark cycle (as opposed to its typicaloccurrence in the light cycle). In certain embodiments, the circadianclock-regulating compound is administered to the mammal in order toincrease the anabolic activity of the liver (e.g., increase the activityof the metabolic pathways of the liver or shift or switch liver activityfrom catabolism to anabolism). In other embodiments, the circadianclock-regulating compound is administered to the mammal in order toincrease the catabolic activity of the liver (e.g., decrease theactivity of the metabolic process).

Mitochondrial Diseases and Metabolic Effects

In addition to regulating circadian rhythms and protect neural cellsfrom cell death, sirtuins such as SIRT3, SIRT4, and SIRT5 are found inmitochondria. SIRT3 is expressed at high levels in metabolically activetissue. Modulation of SIRT3 has a variety of physiological applicationsfor muscle cells including mimicking calorie restriction or exercise,increasing mitochodrial biogenesis or metabolism, sensitizing a cell toglucose uptake, increasing fatty acid oxidation, and decreasing reactiveoxygen species. In addition, SIRT3 is demonstrated herein to be involvedin promoting cell survival during genotoxic stress. Thus modulation ofSIRT3 levels also has applications in mediating cell survival.

Increasing the protein or activity level of SIRT3 in a muscle cell canmimic the benefits of calorie restriction or exercise. In someembodiments, the invention relates to methods for increasingmitochondrial biogenesis or metabolism or for boosting mitochondrialactivity/endurance in a muscle cell by contacting a muscle cell with anagent IS that increases the protein or activity level of SIRT3 in thecell. In some embodiments, the invention relates to methods forsensitizing a muscle cell to glucose uptake by contacting a muscle cellwith an agent that increases the protein or activity level of SIRT3 inthe cell. Further embodiments of the invention relate to methods forincreasing fatty acid oxidation in a muscle cell by contacting a musclecell with an agent that increases the protein or activity level of SIRT3in the cell. Some embodiments of the invention relate to methods fordecreasing reactive oxygen species (ROS) in a muscle cell by contactingthe muscle cell with an agent that increases the protein or activitylevel of SIRT3 in the cell.

Increasing levels of SIRT3 benefits many diseases and disorders affectedby metabolism within mitochondria. Increasing SIRT3 may be useful in anysubjects in need of metabolic activation of one or more of theirmuscles, e.g., smooth muscles or cardiac muscles or muscle cellsthereof. A subject may be a subject having cachexia or muscle wasting.

Increasing SIRT3 may also be used to increase or maintain bodytemperature, e.g., in hypothermic subjects. Alternatively, inhibitingSIRT3 may be used to reduce body temperature, e.g., in subjects havingfever or hyperthermia.

Generally, activation of SIRT3 may be used to stimulate the metabolismof any type of muscle, e.g., muscles of the gut or digestive system, orthe urinary tract, and thereby may be used to control gut motility,e.g., constipation, and incontinence.

Other embodiments in which it would be useful to increase SIRT3 includerepair of muscle, such as after a surgery or an accident, increase ofmuscle mass; and increase of athletic performance.

Thus the invention provides methods in which beneficial effects areproduced by contacting one or more muscle cells with an agent thatincreases the protein or activity level of SIRT3 in the cell. Thesemethods effectively facilitate, increase or stimulate one or more of thefollowing: mimic the benefits of calorie restriction or exercise in themuscle cell, increase mitochondrial biogenesis or metabolism, increasemitochondrial activity and/or endurance in the muscle cell, sensitizethe muscle cell to glucose uptake, increase fatty acid oxidation in themuscle cell, decrease reactive oxygen species (ROS) in the muscle cell,increase PGC-la and/or ucp3 and/or GLUT4 expression in the muscle cell,and activate AMP activated protein kinase (AMPK) in the muscle cell.

Various types of muscle cells can be contacted in accordance with theinvention. In some embodiments, the muscle cell is a skeletal musclecell. In certain embodiments, the muscle cell is a cell of a slow-twitchmuscle, such as a soleus muscle cell. The methods of the inventioninclude, in some embodiments, administering, to a subject in need ofsuch treatment, an agent that increases the protein or activity level ofSIRT3 in cells of the subject.

The cell that is contacted or the subject that is treated in theaforementioned methods preferably is a cell in need of SIRT3 increase inprotein or activity level. In certain embodiments, the cell is adiseased cell of a subject.

Also provided are methods for regulating skeletal muscle metabolism orskeletal muscle energy homeostasis in a subject. In such methods, anagent that modulates the protein or activity level of SIRT3 in thesubject, i.e., the SIRT3 modulators described herein, is administered toa subject in need thereof.

Also provided are methods for increasing the protein level of SIRT3 in amuscle cell or in muscles of a subject. Such methods include subjectinga cell or a subject to caloric restriction or fasting, or administeringto a subject in need thereof an agent that increases the protein oractivity level of SIRT3 in a muscle cell. Diseases, disorders andconditions in which such methods are useful include mitochondrialdiseases, metabolic disorders, neurologic disorders, muscular disorders,cardiovascular diseases, and excessive weight or obesity. Specificmetabolic disorders, diseases or conditions include insulin resistance,diabetes, diabetes related conditions or disorders, endothelialdysfunction, non-alcoholoic fatty liver disease (NAFLD)/non-alcoholichepatic steatosis (NASH), or metabolic syndrome. Other metabolicdisorders will be known to the skilled person.

Mitochondrial diseases that can be treated include diseases that show avariety of symptoms caused by dysfunction of mitochondria in cells. Themitochondrial diseases may be classified in various ways by biochemicalabnormalities, clinical symptoms or types of DNA abnormalities. Typesnamed as KSS (chronic progressive external ophthalmoplegia), MERRF(myoclonus epilepsy associated with ragged-red fibers; Fukuharasyndrome), MELAS, Leber's disease, Leigh encephalopathia and Pearson'sdisease are widely known. Among them, MELAS is a type mainly showingstroke-like episodes, occupies 30% or more of the whole and is believedto be the most frequent type in the mitochondrial disease.

Retinal Diseases and Disorders

Photoreceptor neuronal cell death and vision can be rescued by NMNadministration. In certain embodiments, nicotinamide phosphoribosyltransferase (NAMPT)-mediated NAD biosynthesis can play a role in for rodand/or cone PR neuron survival. In certain embodiments, decreased NADlevels can cause impaired mitochondrial function in PR neurons,alterations in TCA cycle metabolites, and can lead to cell death andblindness.

Deleting NAMPT can lead to photoreceptor death, loss of normal retinalstructure and function, and vision loss. In some cases, damage tophotoreceptor neurons and their function can be reversed withsupplementation of NMN, an NAMPT enzymatic reaction product. Disclosedherein are methods of administering NMN to restore NAD levels in theretina. In some embodiments, NMN supplementation can be an effectivetherapeutic intervention for many retinal degenerative diseases.

Provided herein are methods of treating, preventing, and reducing riskof diseases associated with photoreceptor dysfunction, including,without limitation, age-related macular degeneration (AMD), inheritedand acquired retinal diseases such as, without limitation, retinitispigmentosa (RP), rod and cone dystrophism, and Leber's congenitalamaurosis (LCA) by administration of NMN to a subject. In certainembodiments, NMN administration can be an effective intervention for theprevention and/or treatment of orphan retinal degenerative diseasesincluding but not limited to rod dystrophy, cone dystrophy, retinitispigmentosa, other inherited retinal degenerations, Leber's congenitalamaurosis (LCA) and acquired retinal degenerations such as, but notlimited to, age-related macular degeneration, photoreceptor degenerationfollowing retinal detachment.

In some embodiments, these methods can comprise administering to asubject a pharmaceutically effective amount of nicotinamidemononucleotide (NMN). In some embodiments, a pharmaceutically effectiveamount of nicotinamide mononucleotide (NMN) can be an amount effectivefor increasing retinal NAD levels.

Disclosed herein are methods of treating macular degeneration in asubject. In some embodiments, the methods include treating aberrantretinal NAD levels in a subject, including aberrantly low retinal NADlevels. These methods comprise administering NMN to a subject. In someembodiments, the methods include treating retinal degeneration in asubject. In some embodiments, the methods include treating photoreceptordamage in a subject. In some embodiments, the methods include treatingphotoreceptor degeneration in a subject.

In some embodiments, the methods include treating vision loss associatedwith retinal degeneration in a subject. In some embodiments, the methodsinclude treating aberrant retinal structure in a subject. In someembodiments, the methods include increasing retinal NAD levels in asubject.

In some embodiments, the methods include reducing the risk of developingmacular degeneration in a subject. In some embodiments, the methodsinclude reducing risk of developing aberrant retinal NAD levels in asubject. In some embodiments, the methods include reducing the risk ofdeveloping retinal degeneration in a subject. In some embodiments, themethods include reducing the risk of developing photoreceptordamage/degeneration in a subject. In some embodiments, the methodsinclude reducing the risk of developing vision loss associated withretinal degeneration in a subject. In some embodiments, the methodsinclude reducing the risk of developing aberrant retinal structure in asubject.

In some embodiments, the methods include treating a retina disease in asubject. In some embodiments, a retinal disease that can be treated byadministration of NMN can be retinitis pigmentosa (RP), Leber'scongenital amaurosis (LCA), rod dystrophy, cone dystrophy, rod-conedystrophy, cone-rod dystrophy, age-related macular degeneration,photoreceptor degeneration following retinal detachments, or acombination thereof.

Pharmaceutical Formulations

The compounds of this invention are formulated with conventionalcarriers and excipients, which can be selected in accord with ordinarypractice. Tablets can contain excipients, glidants, fillers, binders andthe like. Aqueous formulations are prepared in sterile form, and whenintended for delivery by other than oral administration generally willbe isotonic. All formulations will optionally contain excipients such asthose set forth in the “Handbook of Pharmaceutical Excipients” (1986).Excipients include ascorbic acid and other antioxidants, chelatingagents such as EDTA, carbohydrates such as dextran,hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid and thelike. The pH of the formulations can range from about 3 to about 11, butis ordinarily about 7 to about 10.

While it is possible for the active ingredients to be administeredalone, it may be preferable to present them as pharmaceuticalformulations. The formulations, both for veterinary and for human use,of the invention comprise at least one active ingredient, as abovedefined, together with one or more acceptable carriers therefor andoptionally other therapeutic ingredients. The carrier(s) must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and physiologically innocuous to the recipientthereof.

The formulations include those suitable for the foregoing administrationroutes. The formulations may conveniently be presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy. Techniques and formulations generally are found in Remington'sPharmaceutical Sciences (Mack Publishing Co., Easton, Pa.). Such methodsinclude the step of bringing into association the active ingredient withthe carrier which constitutes one or more accessory ingredients. Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredient with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tablets,each containing a predetermined amount of the active ingredient as apowder or granules; as a solution or a suspension in an aqueous ornon-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also beadministered as a bolus, electuary or paste.

A tablet is made by compression or molding, optionally with one or moreaccessory ingredients. Compressed tablets may be prepared by compressingin a suitable machine the active ingredient in a free-flowing form suchas a powder or granules, optionally mixed with a binder, lubricant,inert diluent, preservative, surface active or dispersing agent. Moldedtablets may be made by molding in a suitable machine a mixture of thepowdered active ingredient moistened with an inert liquid diluent. Thetablets may optionally be coated or scored and optionally are formulatedso as to provide slow or controlled release of the active ingredienttherefrom.

For infections of the eye or other external tissues, e.g., mouth andskin, the formulations are preferably applied as a topical ointment orcream containing the active ingredient(s) in an amount of, for example,about 0.075 to about 20% w/w (including active ingredient(s) in a rangebetween about 0.1% and about 20% in increments of about 0.1% w/w such asabout 0.6% w/w, about 0.7% w/w, etc.), preferably about 0.2 to about 15%w/w and most preferably about 0.5 to about 10% w/w. When formulated inan ointment, the active ingredients may be employed with either aparaffinic or a water-miscible ointment base. Alternatively, the activeingredients may be formulated in a cream with an oil-in-water creambase.

If desired, the aqueous phase of the cream base may include, forexample, at least about 30% w/w of a polyhydric alcohol, i.e., analcohol having two or more hydroxyl groups such as propylene glycol,butane 1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol(including PEG 400) and mixtures thereof. The topical formulations maydesirably include a compound which enhances absorption or penetration ofthe active ingredient through the skin or other affected areas. Examplesof such dermal penetration enhancers include dimethyl sulphoxide andrelated analogs.

The oily phase of the emulsions of this invention may be constitutedfrom known ingredients in a known manner. While the phase may comprisemerely an emulsifier (otherwise known as an emulgent), it desirablycomprises a mixture of at least one emulsifier with a fat or an oil orwith both a fat and an oil. Preferably, a hydrophilic emulsifier isincluded together with a lipophilic emulsifier which acts as astabilizer. It is also preferred to include both an oil and a fat.Together, the emulsifier(s) with or without stabilizer(s) make up theso-called emulsifying wax, and the wax together with the oil and fatmake up the so-called emulsifying ointment base which forms the oilydispersed phase of the cream formulations.

Emulgents and emulsion stabilizers suitable for use in the formulationof the invention include Tween™ 60, Span™ 80, cetostearyl alcohol,benzyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodiumlauryl sulfate.

The choice of suitable oils or fats for the formulation is based onachieving the desired cosmetic properties. The cream should preferablybe a non-greasy, non-staining and washable product with suitableconsistency to avoid leakage from tubes or other containers. Straight orbranched chain, mono- or dibasic alkyl esters such as di-isoadipate,isocetyl stearate, propylene glycol diester of coconut fatty acids,isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate,2-ethylhexyl palmitate or a blend of branched chain esters known asCrodamol CAP may be used, the last three being preferred esters. Thesemay be used alone or in combination depending on the propertiesrequired. Alternatively, high melting point lipids such as white softparaffin and/or liquid paraffin or other mineral oils are used.

Pharmaceutical formulations according to the present invention comprisea compound according to the invention together with one or morepharmaceutically acceptable carriers or excipients and optionally othertherapeutic agents. Pharmaceutical formulations containing the activeingredient may be in any form suitable for the intended method ofadministration. When intended for oral use for example, tablets,troches, lozenges, aqueous or oil suspensions, dispersible powders orgranules, emulsions, hard or soft capsules, syrups or elixirs may beprepared. Compositions intended for oral use may be prepared accordingto any method known to the art for the manufacture of pharmaceuticalcompositions, and such compositions may contain one or more agentsincluding sweetening agents, flavoring agents, coloring agents andpreserving agents, in order to provide a palatable preparation. Tabletscontaining the active ingredient in admixture with non-toxicpharmaceutically acceptable excipient which are suitable for manufactureof tablets are acceptable. These excipients may be, for example, inertdiluents, such as calcium or sodium carbonate, lactose, calcium orsodium phosphate; granulating and disintegrating agents, such as maizestarch, or alginic acid; binding agents, such as starch, gelatin oracacia; and lubricating agents, such as magnesium stearate, stearic acidor talc. Tablets may be uncoated or may be coated by known techniques,including microencapsulation, to delay disintegration and adsorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate alone or with a wax may be employed.

Formulations for oral use may be also presented as hard gelatin capsuleswhere the active ingredient is mixed with an inert solid diluent, forexample calcium phosphate or kaolin, or as soft gelatin capsules whereinthe active ingredient is mixed with water or an oil medium, such aspeanut oil, liquid paraffin or olive oil.

Aqueous suspensions of the invention contain the active material(s) inadmixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropyl methylcelluose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;and dispersing or wetting agents such as a naturally-occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethyleneoxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol anhydride(e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension mayalso contain one or more preservatives such as ethyl or n-propylp-hydroxy-benzoate, one or more coloring agents, one or more flavoringagents and one or more sweetening agents, such as sucrose or saccharin.

Oil suspensions may be formulated by suspending the active ingredient ina vegetable oil, such as arachis oil, olive oil, sesame oil or coconutoil, or in a mineral oil such as liquid paraffin. The oral suspensionsmay contain a thickening agent, such as beeswax, hard paraffin or cetylalcohol. Sweetening agents, such as those set forth above, and flavoringagents may be added to provide a palatable oral preparation. Thesecompositions may be preserved by the addition of an antioxidant such asascorbic acid.

Dispersible powders and granules of the invention suitable forpreparation of an aqueous suspension by the addition of water providethe active ingredient in admixture with a dispersing or wetting agent, asuspending agent, and one or more preservatives. Suitable dispersing orwetting agents and suspending agents are exemplified by those disclosedabove. Additional excipients, for example sweetening, flavoring andcoloring agents, may also be present.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, suchas olive oil or arachis oil, a mineral oil, such as liquid paraffin, ora mixture of these. Suitable emulsifying agents includenaturally-occurring gums, such as gum acacia and gum tragacanth;naturally-occurring phosphatides, such as soybean lecithin; esters orpartial esters derived from fatty acids; hexitol anhydrides, such assorbitan monooleate; and condensation products of these partial esterswith ethylene oxide, such as polyoxyethylene sorbitan monooleate. Theemulsion may also contain sweetening and flavoring agents. Syrups andelixirs may be formulated with sweetening agents, such as glycerol,sorbitol or sucrose. Such formulations may also contain a demulcent, apreservative, a flavoring or a coloring agent.

The pharmaceutical compositions of the invention may be in the form of asterile injectable preparation, such as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,such as a solution in 1,3-butane-diol or prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile fixed oils may conventionally be employed as a solventor suspending medium. For this purpose, any bland fixed oil may beemployed, including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid may likewise be used in the preparation ofinjectables.

The amount of active ingredient that may be combined with the carriermaterial to produce a single dosage form will vary depending upon thesubject treated and the particular mode of administration. For example,a time-release formulation intended for oral administration to humansmay contain approximately 1 to approximately 1000 mg of active materialcompounded with an appropriate and convenient amount of carrier materialwhich may vary from about 5% to about 95% of the total compositions(weight:weight). The pharmaceutical composition can be prepared toprovide easily measurable amounts for administration. For example, anaqueous solution intended for intravenous infusion may contain fromabout 3 to about 500 μg of the active ingredient per milliliter ofsolution in order that infusion of a suitable volume at a rate of about30 mL/hr can occur.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the activeingredient. The active ingredient is preferably present in suchformulations in a concentration of about 0.5 to about 20%,advantageously about 0.5 to about 10%, and particularly about 1.5% w/w.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for intrapulmonary or nasal administration have aparticle size for example in the range of about 0.1 to about 500microns, such as about 0.5, about 1, about 30, or about 35 microns etc.,which is administered by rapid inhalation through the nasal passage orby inhalation through the mouth so as to reach the alveolar sacs.Suitable formulations include aqueous or oily solutions of the activeingredient. Formulations suitable for aerosol or dry powderadministration may be prepared according to conventional methods and maybe delivered with other therapeutic agents.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

The formulations are presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example water for injection, immediatelyprior to use. Extemporaneous injection solutions and suspensions areprepared from sterile powders, granules and tablets of the kindpreviously described. Preferred unit dosage formulations are thosecontaining a daily dose or unit daily sub-dose, as herein above recited,or an appropriate fraction thereof, of the active ingredient.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavoring agents.

The invention further provides veterinary compositions comprising atleast one active ingredient as above defined together with a veterinarycarrier therefor.

Veterinary carriers are materials useful for the purpose ofadministering the composition and may be solid, liquid or gaseousmaterials which are otherwise inert or acceptable in the veterinary artand are compatible with the active ingredient. These veterinarycompositions may be administered orally, parenterally or by any otherdesired route.

Compounds of the invention are used to provide controlled releasepharmaceutical formulations containing as active ingredient one or morecompounds of the invention (“controlled release formulations”) in whichthe release of the active ingredient are controlled and regulated toallow less frequent dosing or to improve the pharmacokinetic or toxicityprofile of a given active ingredient.

An effective dose of an active ingredient depends at least on the natureof the condition being treated, toxicity, whether the compound is beingused prophylactically (lower doses) or against an active disease ordisorder, the method of delivery, and the pharmaceutical formulation,and will be determined by the clinician using conventional doseescalation studies. It can be expected to be from about 0.0001 to about100 mg/kg body weight per day; typically, from about 0.01 to about 10mg/kg body weight per day; more typically, from about 0.01 to about 5mg/kg body weight per day; most typically, from about 0.05 to about 0.5mg/kg body weight per day. For example, the daily candidate dose for anadult human of approximately 70 kg body weight will range from about 1mg to about 1000 mg, preferably between about 5 mg and about 500 mg, andmay take the form of single or multiple doses.

Routes of Administration

One or more compounds of the invention (herein referred to as the activeingredients) are administered by any route appropriate to the conditionto be treated. Suitable routes include oral, rectal, nasal, topical(including buccal and sublingual), vaginal and parenteral (includingsubcutaneous, intramuscular, intravenous, intradermal, intrathecal andepidural), and the like. It will be appreciated that the preferred routemay vary with, for example, the condition of the recipient. An advantageof the compounds of this invention is that they are orally bioavailableand can be dosed orally.

While the foregoing written description of the invention enables one ofordinary skill to make and use what is considered presently to be thebest mode thereof, those of ordinary skill will understand andappreciate the existence of variations, combinations, and equivalents ofthe specific embodiment, method, and examples herein. The inventionshould therefore not be limited by the above described embodiment,method, and examples, but by all embodiments and methods within thescope and spirit of the invention.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe following specification and attached claims are approximations thatmay vary depending upon the desired properties sought to be obtained bythe present invention. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits and by applying ordinaryrounding techniques.

It is to be understood that wherever values and ranges are providedherein, all values and ranges encompassed by these values and ranges,are meant to be encompassed within the scope of the present invention.Moreover, all values that fall within these ranges, as well as the upperor lower limits of a range of values, are also contemplated by thepresent application.

INCORPORATION BY REFERENCE

All U.S. patents and U.S. and PCT published patent applications andnon-patent literature mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

EXAMPLES

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents were considered to be within the scope of thisinvention and covered by the claims appended hereto. For example, itshould be understood, that modifications in reaction conditions,including but not limited to reaction times, reaction size/volume, andexperimental reagents, such as solvents, catalysts, pressures,atmospheric conditions, e.g., nitrogen atmosphere, andreducing/oxidizing agents, with art-recognized alternatives and using nomore than routine experimentation, are within the scope of the presentapplication.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the compounds and methods of the invention, and are notintended to limit the scope of what the inventor(s) regard(s) as theinvention.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

Unless noted otherwise, the starting materials for the synthesisdescribed herein were obtained from commercial sources or knownsynthetic procedures and were used without further purification.

Analytical Methods

LC/MS data were obtained on an Agilent 1260 Infinity System, equippedwith a Model 1260 ELSD and DAD and a Model 6120 Quadrupole MassDetector. The HPLC analyses were run using either a Waters Atlantis C₁₈(100×4.6 mm, 3μ 100 Å) using a gradient of 10 mM NH₄OAc and methanol, oran Agilent Poroshell EC-C₁₈ (4.6×50 mm, 2.7μ) using a gradient of 0.1%formic acid in water and acetonitrile. NMR Spectra were obtained on aBruker BioSpin 500 MHz Avance III Digital NMR spectrometer. Protonspectra are reported in ppm and are relative to TMS and ³¹P spectra (122MHz) are relative to 85% orthophosphoric acid as an external reference.Materials were obtained from commercial sources and were used withoutpurification. All solvents were either HPLC-grade or anhydrous, asindicated below.

Example 1:1-((2R,3R,4S)-5-(((4-(3-Chlorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-1λ⁴-pyridine-3-carboxamideTrifluoroacetate salt (Compound 1) Example 1A:(3-carbamoyl-1-(6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pyridin-1-iumtriflate salt)

A 500 mL 3-necked flask, under nitrogen, was charged with 110 mL ACN.The solvent was cooled on ice to 0-5° C. and treated with 1.2 mLsulfuric acid. After 5 minutes, the solution was treated with 28 gm (269mmol) 2,2-dimethoxypropane. Next, the solution was treated with 13.65 gm(33.7 mmol) nicotinamide riboside triflate salt (Sauve et al., WO2007/061798) and the reaction was allowed to warm to room temperatureand monitored by LC. After 20 minutes, the dark solution was cooled onan ice-water bath and treated with 2.73 gm (25.7 mmol) sodium carbonateand 5 mL water. After stirring for 20 minutes, the mixture was filteredto remove any remaining solid carbonate, and the filtrate evaporated toafford 19 gm as a dark red foam. The bulk sample was dissolved in 200 mLMeOH and treated, while stirred, with 50 mL water. At thisconcentration, the completely dissolved material became a bit turbid.The mixture was treated with 45 gm charcoal and allowed to stand at roomtemperature for 2 hours. The mixture was filtered through Celite, whichwas washed with MeOH and the filtrate evaporated to produce a turbidliquid. The liquid was diluted with ˜30 mL ACN until a clear solutionwas obtained, which was frozen and lyophilized to afford 11.4 gm3-carbamoyl-1-(6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pyridin-1-iumtriflate salt, as a pale yellow solid (76%).

¹H NMR (CDCl₃): δ 9.2 (s, 1H); 8.9 (d, 1H); 8.7 (d, 1H); 8.0 (q, 1H);6.0 (d, 1H); 4.9 (dd, 1H); 4.7 (d, 1H); 4.6 (bd s, 1H); 3.8 (dd, 1H);3.6 (dd, 1H); 1.4 (s, 3H); 1.2 (s, 3H).

Example 1B:1-((3aR,4R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamide

A 3 L flask was charged with a solution of 55 gm (124 mmol)3-carbamoyl-1-(6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pyridin-1-iumin 1 L DCM. The solution was treated with treated with 1.5 L H₂O and wasdegassed by bubbling argon though it for 20 minutes. The vigorouslystirred mixture was cooled on an ice bath and treated with 52 gm (618mmol) NaHCO₃, followed by 108 gm (618 mmol) sodium dithionite added inportions to control the foaming.

After the addition was complete, the mixture was allowed to warm to roomtemperature. After three hours, an additional 20 gm sodium dithionitewas added and the reaction was stirred overnight. The two layers wereseparated and the aqueous layer was extracted with 250 mL DCM (2×). Thecombined DCM layer was dried over sodium sulfate, filtered and strippedto provide 27.3 gm of crude product. The crude material was purified viaflash chromatography with a gradient of 0-6% MeOH in DCM. The pooledproduct fractions were evaporated to yield 9.1 gm1-((3aR,4R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamideas a pale solid.

¹H NMR (CDCl₃): δ 7.2 (d, 1H); 6.0 (dd, 1H); 5.6 (bd s, 2H); 4.9 (dd,1H); 4.8 (m, 2H); 4.6 (dd, 1H); 1.6 (s, 3H); 1.4 (s, 3H).

Example 1C:1-((3aR,4R,6aR)-6-(((4-(3-chlorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamide

A 250 mL round bottom flask was placed under an inert atmosphere withargon and charged with 2 gm (6.75 mmol)1-((3aR,4R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamide.Under argon, the acetonide was dissolved in 75 mL dry DMF and treatedwith 10.1 mL tert-butylmagnesium chloride (10.1 mmol; 1M in THF) andstirred for 30 minutes. The reaction was treated with a solution of 2.74gm (7.42 mmol)(±)-4-(3-chlorophenyl)-2-(4-nitrophenoxy)-1,3,2-dioxaphosphinane 2-oxide(Erion, et al., JACS, 126, 5154 (2004)) in 15 mL anhydrous DMF and thereaction warmed to 45 C and stirred. The reaction was monitored by LCand was complete within 3.5 hours. The dark solution was cooled to roomtemperature, stripped, co-evaporated with 2×ACN and dried on high vacuumto give 7.3 gm as a dark semi-solid. This product was purified via flashchromatography on silica with a gradient of 0-10% MeOH in DCM. Pooledfractions were evaporated to dryness on high vacuum to give 1.51 gm1-((3aR,4R,6aR)-6-(((4-(3-chlorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamideas a pale yellow solid (42%).

¹HNMR (CDCl₃): δ 7.3 (m, 4H); 7.1 (s, 1H); 5.8 (t, 1H); 4.8 (t, 1H); 4.4(m, 3H); 4.1 (m, 1H); 3.1 (d, 1H); 2.1 (m, 2H); 1.5 (s, 3H); 1.3 (s,3H).

³¹P (CDCl₃): −4.1 and −4.4 ppm.

MS (ES-API⁺) m/z M=526 (M+)

Example 1D:1-((3aR,4R,6aR)-6-(((4-(3-chlorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1λ⁴-pyridine-3-carboxamide

A 250 mL flask was charged with 1.5 gm (2.8 mmol)1-((3aR,4R,6aR)-6-(((4-(3-chlorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamideand dissolved in 60 mL MeOH. The solution was treated with 709 mg (2.8mmol) CoAc₂-4H₂O and stirred to dissolve. The resulting solution wastreated with 1.0 mL 30% aqueous H₂O₂ and the mixture was stirred at roomtemperature. The reaction was monitored by HPLC and was virtuallycomplete after 90 minutes. The mixture was treated with an additional 50μL hydrogen peroxide and the reaction was complete after 3.5 hours. Thesolution was treated with 7.5 gm QuadraSil AP resin and 5 mL water andstirred at room temperature for 45 minutes. The resin was filtered andwashed with 75 mL 2:1 MeOH—H₂O. The filtrate was stripped to remove mostof the MeOH, frozen and lyophilized to give 1.44 gm1-((3aR,4R,6aR)-6-(((4-(3-chlorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1λ⁴-pyridine-3-carboxamideas a dark semi-solid. The crude material is used directly in the nextreaction. MS (ES-API⁺) m/z=526 (M+)

Example 1E:1-((2R,3R,4S)-5-(((4-(3-chlorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-1λ⁴-pyridine-3-carboxamideTrifluoroacetate salt (Compound 1)

A 100 mL flask was charged with a solution of 1.44 gm1-((3aR,4R,6aR)-6-(((4-(3-chlorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1λ⁴-pyridine-3-carboxamideacetate (2.45 mmol, 1×) in 15 mL DCM. This solution was treated with 15mL 90% TFA/H₂O and the resulting solution stirred at 35° C. The reactionwas complete after 3 hrs. The solvent was stripped and the residue isco-evaporated from 2×10 mL acetonitrile and then dried on high vacuum,to afford 1.83 gm, as a dark oil. This oil was purified on silica using10% MeOH in DCM, containing 1% formic acid and 2% water.

The pooled product fractions were stripped and co-evaporated from 2×5 mLwater to remove any residual formic acid. The residue was frozen andlyophilized to give 769 mg1-((2R,3R,4S)-5-(((4-(3-chlorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-1λ⁴-pyridine-3-carboxamideas the trifluoroacetic acid salt, 769 mg as a tan solid (45% 2-stepyield). LC shows two peaks corresponding to the diastereomers.

¹H NMR (ACN-d₃ and D₂O): δ 9.3 (m, 1H); 9.1 (m, 1H); 8.9 (m, 1H); 7.3(m, 4H); 6.1 (m, 1H); 5.6 (m, 1H); 4.6-4.4 (m, 5H); 2.2-1.9 (m, 2H).

³¹P (ACN-d₃ and D₂O): −3.6 and −5.3 ppm.

MS (ES-API⁺) m/z=485 (M+).

Example 2:1-((2R,3R,4S)-5-(((4-(3,5-Difluorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-1λ⁴-pyridine-3-carboxamideTrifluoroacetate salt (Compound 2) Example 2A:1-((3aR,4R,6aR)-6-(((4-(3,5-difluorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamide

A 250 mL round bottom mask was charged with 1.1 gm (3.71 mmol)1-((3aR,4R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamideand flushed with nitrogen. The acetonide was dissolved in 40 mL dry DMFand treated with 5.57 mL (5.57 mmol) tert-butylmagnesium chloride (1M inMeTHF) and stirred for 30 minutes, then treated with a solution of 1.59gm (4.08 mmol)(±)-4-(3,5-difluorophenyl)-2-(4-nitrophenoxy)-1,3,2-dioxaphosphinane2-oxide (Erion, et al., JACS, 126, 5154 (2004)) in 10 mL dry DMF. Thesolution was warmed to 45° C. and stirred for two hours and then at roomtemperature overnight. The solvent was stripped to afford an oil that istaken up in ACN and co-evaporated (3×25 mL). The residue is placed onhigh vacuum to give 3.73 gm1-((3aR,4R,6aR)-6-(((4-(3,5-difluorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamideas an orange solid.

¹H NMR (ACN-d₃): δ 7.0-6.9 (m, 3H); 6.9 (m, 1H); 6.0 (m, 1H); 5.6 (m,1H); 4.9 (m, 1H); 4.75 (m, 1H); 4.70 (m, 1H); 4.3 (m, 2H); 4.07 (m, 1H);3.0 (m, 2H); 2.3-2.2 (m, 2H); 1.5 (s, 3H); 1.3 (s, 3H).

³¹P NMR (ACN-d₃): 4.3 and −4.5 ppm

MS (ES-API⁺) m/z=529 (M+H⁺).

Example 2B:1-((3aR,4R,6aR)-6-(((4-(3,5-Difluorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1λ⁴-pyridine-3-carboxamideacetate salt

A 250 mL flask was charged 1.25 gm (2.36 mmol)1-((3aR,4R,6aR)-6-(((4-(3,5-difluorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamideand was dissolved in a solution of 589 mg (2.36 mmol) CoAc₂-4H₂O in 55mL MeOH and cooled on an ice bath. The resulting solution is treatedwith 300 μL 30% aqueous H₂O₂ and the mixture stirred at 0° C. and thenallowed to warm to room temperature and the reaction monitored by HPLC.After progressing ˜30% in 2 hours, the dark solution was treated with anadditional 300 μL peroxide, followed by another 325 μL over the nexthour. The reaction is treated with 5 gm Quadrapure AP silica captureresin that is suspended in 10 mL MeOH, and 10 mL water and stirred for45 minutes. The resin is removed by filtration and rinsed with MeOH andwater, and the filtrate was frozen and lyophilized to give 1.36 gm1-((3aR,4R,6aR)-6-(((4-(3,5-difluorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1λ⁴-pyridine-3-carboxamideacetate salt as a dark solid. The crude product was used directly in thenext reaction.

Example 2C:1-((2R,3R,4S)-5-(((4-(3,5-Difluorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-1λ⁴-pyridine-3-carboxamideTrifluoroacetate salt (Compound 2)

A 100 mL flask was charged with 1.38 gm (2.3 mmol)1-((3aR,4R,6aR)-6-(((4-(3,5-difluorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1λ⁴-pyridine-3-carboxamideacetate salt and dissolved 20 mL DCM. The solution was treated with 20mL 90% TFA/10% H₂O and stirred at 35° C. for 90 minutes. The solvent isremoved in vacuo and the residue co-evaporated from ACN (2×), then driedunder high vacuum to give 1.78 gm as a dark solid. The solid waspurified via flash chromatography using 15% MeOH in DCM, containing 2%water and 1% formic acid. The pooled product fractions were stripped toan oil and co-evaporated from water (2×) to remove residual formic acid.The product was frozen and lyophilized to give 811 mg1-((2R,3R,4S)-5-(((4-(3,5-difluorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-1-pyridine-3-carboxamidetrifluoroacetate salt as an off-white solid (58%).

¹H NMR (D₂O): δ 9.4 (d, 1H); 9.2 (t, 1H); 8.9 (dd, 1H); 8.2 (m, 1H); 7.0(m, 3H); 6.25 (m, 1H); 5.7 (dd, 1H); 4.7 (m, 3H); 4.5 (m, 2H); 4.4 (m,1H); 2.4 (m, 2H).

³¹P NMR (D₂O): −3.8 and −4.1 ppm.

MS (ES-API⁺) m/z=487 (M+)

Example 3:S-(1-((((5-(3-carbamoylpyridin-1(4H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(2-(pivaloylthio)ethoxy)phosphoryl)oxy)ethan-2-yl)2,2-dimethylpropanethioate Trifluoroacetate salt (Compound 3) Example3A: S-(2-Hydroxethyl) thiopivaloate

This compound was prepared in 86% yield according to the procedure ofLefebvre et al. J. Med. Chem. 1995, 38, 3941-3950.

Example 3B: Bis(S-pivaloyl-2-thioethyl) N,N-diisopropylphosphoramidite

This compound was prepared according to the procedure of Lefebvre et al,J Med. Chem. 1995, 38, 3941-3950. (89.6%)

Example 3C: Bis SATE (P3)NMNH Acetonide:S,S-(((((6-(3-carbamoylpyridin-1(4H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)phosphanediyl)bis(oxy))bis(ethane-2,1-diyl))bis(2,2-dimethylpropanethioate)

1-((3aR,4R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamide,prepared according to Example 1B, (0.541 g, 1.82 mmoles) in a dry 25 mlone necked round bottomed flask. Dry ACN (1 ml) was added, the solutionwas stirred briefly and then evaporated to provide a yellow foam. DryACN (5 ml) was added, stirred, then the bis(S-pivaloyl-2-thioethyl)N,N-diisopropylphosphoramidite (850 mg, 1.88 mmoles) was added. Thissolution was cooled to −20° C. for 5 min, then tetrazole in ACN (0.45molar, 2.71 ml, 1.22 mmoles) was added dropwise. The cold bath wasremoved and the reaction allowed to warm to RT, and allowed to reactovernight. Another 0.7 ml of the tetrazole solution was added and thereaction given another 1.5 hr at RT. The solvent was removed in vacuo.Saturated degassed aqueous NaHCO₃ (5 ml) and dichloromethane (5 ml)solutions were added. The solution was stirred vigorously, then allowedto separate. The DCM phase was dried (Na₂SO₄), filtered and concentratedin vacuo to give 0.610 g of a yellow glass. 52% yield.

¹H NMR (CDCl₃) ppm 7.13 (1H, m), 6.01 (1H, m), 5.30 (bs, 2H), 4.88 (m,2H), 4.71 (m, 1H), 4.65 (m, 1H), 4.14 (m, 1H), 4.05-3.9 (m, 6H),3.15-3.0 (m, 4H), 1.58 (s, 3H), 1.37 (s, 3H)

³¹P (CD₃OD) 140.02 ppm.

MS(ESI+) m/z=649 (M+H)

Example 3D: Bis SATE ester (P5) NMNH Acetonide:S,S-(((((6-(3-carbamoylpyridin-1(4H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)phosphordiyl)bis(oxy))bis(ethane-2,1-diyl))bis(2,2-dimethylpropanethioate)

The Bis SATE (P3) NMNH Acetonide (2.19 gm, 3.38 mmoles) was dissolved indry MeOH (7 ml) in a 50 ml 14/20 one necked round bottomed flask with amagnetic stir bar. The flask was placed into an ice/water bath andallowed to cool for 5 minutes. Hydrogen peroxide (30% solution in water)(325 μl, 358 mg, 3.18 mmoles, 0.94 eq) was added and the reactionmonitored by TLC (5% MeOH/DCM). The reaction was concentrated in vacuo,dissolved in DCM, dried over Na₂SO₄, filtered and concentrated to give2.71 g of a glass. This was dissolved in DCM and purified on 15 gms ofsilica gel (Baker), eluting with DCM (100 ml), 1% MeOH/DCM (200 ml) and3% MeOH/DCM (200 ml). The appropriate fractions were collected to give1.42 g (63%) of product.

¹H NMR (CDCl₃) 7.08 (bs, 1H), 5.90 (m, 1H), 5.33 (bs, 2H), 4.89-4.71 (m,2H), 4.71-4.68 (m, 1H), 4.68-4.62 (m, 1H), 4.30-4.10 (325, 7H),3.20-3.10 (mg, 6H), 1.56 (bs, 3H), 1.36 (bs, 3H), 1.25 (bs, 18H).³¹P(CD₃OD) −1.01 ppm.

MS(ESI+) m/z=665 (M+H⁺)

Example 3E: Bis SATE ester NMN Acetonide Acetate Salt:1-(6-(((bis(2-(pivaloylthio)ethoxy)phosphoryl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-3-carbamoylpyridin-1-iumacetate salt

The Bis SATE ester (P5) NRH Acetonide (1.40 g, 2.10 mmoles) was placedinto a 100 ml 1 necked round bottomed flask with a magnetic stir bar andplaced under N₂. Dry MeOH (6 ml) was added via syringe to give a yellowsolution. Cobalt acetate tetrahydrate (0.525 g, 2.10 mmoles) was addedand the reaction was stirred to give burgundy solution. The solution wascooled in an ice/water bath, then hydrogen peroxide (30% solution, 895mg, 8.69 mmoles, 4.14 eq) was added gradually until TLC (5% MeOH/DCM)showed complete reaction. Upon addition of the H₂O₂, the reaction becameolive green in color. The reaction was concentrated in vacuo anddissolved in DCM. This solution was washed with water (a little sat'dNaCl solution was used to help the layers form). The DCM phase was driedover Na₂SO₄, filtered and concentrated to a green foam (1.41 g). HPLCconfirmed conversion of the starting material to a new entity, MSconfirmed the identity.

MS(ESI+) m/z=663(M+)

This material was used directly in the Example 3F reaction.

Example 3F: Bis SATE ester NMN Trifluoroacetate Salt:1-(5-(((bis(2-(pivaloylthio)ethoxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-3-carbamoylpyridin-1-iumTrifluoroacetate salt (Compound 3)

The Bis SATE ester NMN Acetonide Acetate Salt (1.41 g, 1.95 mmoles) wasdissolved in 15.6 ml of trifluoroacetic acid, 17 ml of dichloromethaneand 1.4 ml of water and stirred at 35° C. for 1.5 hrs. The mixture wasconcentrated in vacuo and co-evaporated with acetonitrile (3×). Theresulting glass was dissolved in DCM and placed onto a 10 g silica gelcolumn and eluted with DCM (100 ml), 1% MeOH/DCM (150 ml), 3% MeOH/DCM(100 ml), 10% MeOH/DCM (150 ml) then 30% MeOH/DCM (200 ml). This gave810 mg of a colored glass which was dissolved in MeOH (+a little water)and treated with Quadra-Sil resin (5 gm) and stirred for 15 min.Filtration and washing of the resin with MeOH gave a filtrate that wasconcentrated in vacuo to give 758 mg of a tea colored glass.

¹H NMR (D₂O): ppm 9.38 (bs, 1H), 9.14 (bd, 1H), 8.95 (bs, 1H), 8.25 (bt,1H), 6.21 (bd, 1H), 4.54 (m, 1H), 4.42-4.29 (m, 2H), 4.28-4.25 (m, 1H),4.13-4.04 (bq, 4H), 1.08 (bs, 18H).

³¹P (D₂O): 0.76 (s) ppm.

MS(ESI+) m/z=623.7 (M⁺).

Example 4: Isopropyl((((3S,4R,5R)-5-(3-carbamoyl-1λ⁴-pyridin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninateTrifluoroacetate salt (Compound 4) Example 4A: Isopropyl((((3aR,6R,6aR)-6-(3-carbamoylpyridin-1(4H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate

A 100 mL round bottom flask vial was charged with 1.1 gm (3.71 mmol)1-((3aR,4R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamideand flushed with nitrogen. The compound was dissolved in 35 mL dry THFand treated with 4.45 mL (4.45 mmol) tert-butylmagnesium chloride (SAF;1M in THF)) and the solution stirred for 30 minutes. The solution wastreated via syringe with a solution of 1.36 gm isopropyl(chloro(phenoxy)phosphoryl)-L-alaninate (McGuigan, et al., J. Med.Chem., 48, 3504 (2005)) in 20 mL dry THF over a few minutes and thereaction stirred at room temperature. The reaction was allowed to stirovernight at ambient temperature. The reaction mixture was treated with˜15 mL saturated NH₄Cl and stripped to an oil, and the residue taken upin DCM, washed with brine and the brine back-extracted with DCM (2×).The combined organics were dried over sodium sulfate, filtered andevaporated to dryness to give 1.92 gm as a yellow foam. The foam waspurified via flash chromatography with 2% MeOH in DCM. The pooledproduct fractions were evaporated to give 1.16 gm isopropyl((((3aR,6R,6aR)-6-(3-carbamoylpyridin-1(4H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(phenoxy)phosphoryl)-L-alaninateas a bright yellow foam (53%).

¹H NMR (CDCl₃): δ 7.3 (m, 6H); 5.8 (m, 1H); 5.6 (s, 1H); 5.0 (m, 1H);4.8 (d, 1H); 4.7 (m, 2H); 4.6 (m, 2H); 4.0 (m, 1H); 3.1 (m, 2H); 1.5, s,3H); 1.4 (d, 3H); 1.3 (s, 3H); 1.2 (d, 6H).

³¹P (CDCl₃): 3.6 ppm.

MS (ES-API⁺) m/z=566 (M+H⁺)

Example 4B: Isopropyl((((3aR,6R,6aR)-6-(3-carbamoyl-1λ4-pyridin-1-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(phenoxy)phosphoryl)-L-alaninateacetate salt

This synthesis follows the procedure described in Monatsh für Chemie,134:107 (2003). A 100 mL round bottomed flask was charged with 484 mg(1.94 mmol) Co(Ac)₂—(H₂O)₄ which was dissolved in 50 mL MeOH. The redsolution was cooled to 0° C. and treated with 1.1 gm (1.94 mmol)isopropyl((((3aR,6R,6aR)-6-(3-carbamoylpyridin-1(4H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate.The resulting solution is treated with 500 μL 30% aqueous H₂O₂ and themixture stirred at room temperature. After 2 hours, an additional 20 μLperoxide was added. The reaction was complete after a total of 4 hours.The reaction was treated with 5 gm QuadraSil AP resin suspended in 10 mLMeOH and 10 mL water and stirred at room temperature for 20 minutes,whereupon the solid was filtered and washed with MeOH and water. Thefiltrate was stripped to remove the MeOH and the resulting solution wasfrozen and lyophilized to give 1.12 gm isopropyl((((3aR,6R,6aR)-6-(3-carbamoyl-1λ⁴-pyridin-1-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(phenoxy)phosphoryl)-L-alaninateacetate salt as a dark solid. The crude product is used directly in theExample 4C reaction.

MS (ES-API⁺) m/z=564 (M⁺).

Example 4C: Isopropyl((((3S,4R,5R)-5-(3-carbamoyl-1λ4-pyridin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninateTrifluoroacetate salt (Compound 4)

A 250 mL flask was charged with 1.1 gm isopropyl((((3aR,6R,6aR)-6-(3-carbamoyl-1λ⁴-pyridin-1-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate as the acetate salt (1.8 mmol). This materialwas dissolved 20 mL DCM, treated with 20 mL 90% TFA/H₂O and theresulting solution stirred at 35° C. for 90 minutes. The reaction wasstripped to an oil and the residue co-evaporated with ACN (3×) to give 2gm of red-brown oil. Purified via flash chromatography with 15% MeOH/DCMcontaining 1% formic acid and 2% water. Product fractions were pooled,stripped to an oil and then co-evaporated with water (2×). The remainingwater was frozen and lyophilized to give 619 mg isopropyl((((3S,4R,5R)-5-(3-carbamoyl-1λ⁴-pyridin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninatetrifluoroacetateas an orange solid (54%).

¹H NMR (D₂O): δ 9.3 (s, 1H); 9.1 (s, 1H); 8.7 (s, 1H); 8.2 (s, 1H);7.3-7.0 (m, 5H); 6.2 (d, 1H); 4.9 (m, 1H); 4.7 (m, 1H); 4.6 (m, 1H); 4.4(m, 1H); 4.2 (m, 1H); 3.9 (m, 1H); 1.6 (d, 3H); 1.2 (d, 6H).

³¹P NMR (D₂O): δ 5.4 and 5.3 ppm, in a ratio of ˜2:1.

MS (ES-API⁺) m/z=526.3. (M+H⁺)

Example 5: Neopentyl((((3S,4R,5R)-5-(3-carbamoyl-1λ4-pyridin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(naphthalen-1-yloxy)phosphoryl)alaninateTrifluoroacetate salt (Compound 5) Example 5A: Neopentyl((((3aR,6R,6aR)-6-(3-carbamoylpyridin-1(4H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(naphthalen-1-yloxy)phosphoryl)alaninate

A 250 mL flask was charged with 1.73 gm (5.82 mmol)1-((3aR,4R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamideand the flask flushed with nitrogen. This material was dissolved in 50dry DMF and treated with 6.99 mL (6.99 mmol) tert-butylmagnesiumchloride (1M in THF)). The solution was stirred for 30 minutes at roomtemperature, at which time it was treated with a solution of 3.4 gm(6.99 mmol) neopentyl((naphthalen-1-yloxy)(4-nitrophenoxy)phosphoryl)alaninate (Eneroth, etal., US Patent Pub. No. 2013/0143835) in 25 mL dry DMF and the reactionwas stirred at room temperature. The solvent was removed in vacuo andthe residue co-evaporated from ACN (2×). The product was purified viaflash chromatography with a gradient of 0-15% MeOH in DCM. The productfractions were pooled and evaporated on high vacuum to give 2.62 gmneopentyl((((3aR,6R,6aR)-6-(3-carbamoylpyridin-1(4H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(naphthalen-1-yloxy)phosphoryl)alaninate.

¹HNMR (CDCl₃): δ 8.2-7.2 (8H); 5.7 (m, 1H); 4.9 (m, 1H); 4.5-4.1 (5H);3.7-3.5 (4H); 3.0 (dd, 2H); 1.4-0.9 (18H).

³¹P (CDCl₃): 4.9 and 4.2 ppm.

MS (ES-API⁺) m/z=644 (M+).

Example 5B: Neopentyl((((3aR,6R,6aR)-6-(3-carbamoyl-1λ4-pyridin-1-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(naphthalen-1-yloxy)phosphoryl)alaninateacetate salt

A 250 mL flask was charged with 2.6 gm (4 mmol) neopentyl((((3aR,6R,6aR)-6-(3-carbamoylpyridin-1(4H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(naphthalen-1-yloxy)phosphoryl)alaninateand the compound was dissolved in 100 mL MeOH. The solution was treatedwith 1.0 gm (4.0 mmol) CoAc₂-4H₂O in 20 mL MeOH, followed by 2 mL 30%H₂O₂ and the dark the mixture was allowed to stir at room temperature.The reaction was monitored by LC and was ˜80% complete after two hours.The mixture was treated with an additional 500 μL peroxide and wascomplete within 30 minutes. The solution was treated with 13 gmQuadraSil AP resin and ˜10 mL water and stirred for 1 hour. The resinwas removed by filtration and washed with MeOH. The filtrate wasconcentrated on high vacuum to afford neopentyl((((3aR,6R,6aR)-6-(3-carbamoyl-1λ⁴-pyridin-1-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(naphthalen-1-yloxy)phosphoryl)alaninateacetate salt as a dark solid. The crude material was used directly inthe Example 5C reaction.

MS (ES-API⁺) m/z=642 (M+).

Example 5C: Neopentyl((((3S,4R,5R)-5-(3-carbamoyl-1λ4-pyridin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(naphthalen-1-yloxy)phosphoryl)alaninateTrifluoroacetate salt (Compound 5)

A 250 mL flask was charged with crude neopentyl((((3aR,6R,6aR)-6-(3-carbamoyl-1λ⁴-pyridin-1-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(naphthalen-1-yloxy)phosphoryl)alaninateacetate salt from the Example 5B reaction and it was dissolved in 25 mLDCM. The resulting solution is treated with 25 mL 90% TFA/H₂O andstirred at 37° C. for 45 minutes. The solvent was stripped on highvacuum and the residue was co-evaporated with ACN (2×), diluted withwater, frozen and lyophilized to give 3.5 gm of a dark oil. The oil waspurified via flash chromatography with a gradient of 5-15% MeOH in DCM,containing 1% HCO₂H-2% H₂O. The pooled product fractions were strippedand then co-evaporated with water (3×) to remove the residual formicacid. The material was placed on high vacuum and evaporated to give,1.42 gm neopentyl((((3S,4R,5R)-5-(3-carbamoyl-1λ⁴-pyridin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(naphthalen-1-yloxy)phosphoryl)alaninatetrifluoroacetate salt as a tan solid (49% 2-step yield).

¹H NMR (ACN-d₃) (For some of the proton signals, the diastereomers wereclearly differentiated, as noted): δ 9.7 and 9.5 (bs s, 1H); 9.1-7.3(12H); 6.1 and 6.0 (m, 1H); 4.9 (m, 1H); 4.5 (m, 2H); 4.3 and 4.2 (m,2H); 3.9 (m, 1H); 3.7 (m, 2H); 1.4 and 1.3 (d, 3H); 0.9 and 0.8 (s, 9H).

³¹P NMR (ACN-d₃) (Two diastereomers): 5.8 and 5.6 ppm.

MS (ES-API⁺) m/z=602 (M+).

Example 6:1-(5-((((Benzylamino)(2-(pivaloylthio)ethoxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-3-carbamoylpyridin-1-iumTrifluoroacetate salt (Compound 6) Example 6A:S-(2-(((Benzylamino)(4-nitrophenoxy)phosphoryl)oxy)ethyl)2,2-dimethylpropanethioate

4-Nitrophenylphosphoro dichloride (2.52 g, 9.86 mmoles) was placed intoa dried 100 ml single necked round bottomed flask and placed underargon. Anhydrous THF (15 ml) was added and the solution degassed andstirred, and placed under argon. The solution was placed into a dryice/acetone bath and cooled to −78° C. Triethylamine (4.12 ml, 2.99 g,29.6 mmols) was added dropwise to the cold solution. S-(2-hydroxyethyl)2,2-dimethylpropanethioate (1.60 g, 9.86 mmoles) was then added dropwiseto the cold reaction mixture. It was removed from the cold bath andallowed to warm to room temperature. After 30 minutes at roomtemperature, the reaction was again cooled to −78° C. and benzyl amine(1.07 ml, 1.06 g, 9.86 mmoles) was added dropwise. The cold bath wasremoved and the reaction allowed to warm to room temperature. After 1hour at room temperature, the solvent was removed in vacuo and ethylacetate (15 ml) was added. The resulting solution was filtered, thesolid was washed with ethyl acetate (10 ml) and the filtrate was washedwith water (20 ml). The layers were separated and the organic phasewashed with saturated NaCl solution (10 ml) then dried over Na₂SO₄. Theorganic phase was filtered and concentrated in vacuo. The resulting oilwas dissolved in dichloromethane (10 ml) and silica gel (2 g) was addedto the solution. It was then filtered and the silica gel washed withmore dichloromethane (20 ml). The combined solution was concentrated invacuo to give 3.81 g (86%) of product, which was used in the nextreaction.

¹HNMR (CDCl₃) ppm 8.22 (d, 2H), 7.40-7.30 (bm, 7H), 5.33 (bs, 1H),4.28-4.16 (m, 4H), 3.85-3.65 (m, 1H), 3.25-3.14 (bt, 2H), 1.26 (bs, 3H).

³¹P (CDCl₃) 4.52 ppm(s).

Example 6B:S-(2-(((Benzylamino)((6-(3-carbamoylpyridin-1(4H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)phosphoryl)oxy)ethyl)2,2-dimethylpropanethioate

1-(6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamide(2.48 g, 8.36 mmoles) was placed in a dry 100 ml single necked roundbottomed flask and put under N₂. Anhydrous DMF (4 ml) was added viasyringe and this gave a yellow solution. This solution was evacuated for15 min to remove volatiles and traces of water, then another portion ofdry DMF (11 ml) was added via syringe. This solution was placed into anice/water cold bath for 15 min. A 1M solution of t-butyl magnesiumchloride in THF (10 ml, 10 mmols 1.2 eq) was added dropwise to thecooled solution over a 35 min period. During the addition a solid formedin the reaction. A 1 ml portion of dry DMF was added to the reactionmixture to form a homogeneous solution. This solution was stirred coldfor 1.5 hrs, and gave a yellow, heterogeneous solution.S,S-((((4-nitrophenoxy)phosphordiyl) bis(oxy))bis(ethane-2,1-diyl))bis(2,2-dimethylpropanethioate) (3.50 g, 7.74 mmoles, 0.93 eq) wasdissolved in dry DMF (3 ml) and added dropwise to the cold anionsolution over 10 min. The reaction became orange and homogeneous. It wasremoved from the cold bath and allowed to warm to room temperature, andafter 30 min, the reaction was cloudy and had a green tint. The reactionwas allowed to stir at room temperature for 2 hours, then concentratedin vacuo. The resulting glass was dissolved in dichloromethane andloaded onto 60 Silica gel prepared in dichloromethane. The elutionsolvent was as follows: DCM (150 ml), 5% MeOH/DCM (200 ml) then 10%MeOH/DCM (300 ml). This gave 3.00 g of product in 63.6% yield.

¹H NMR (CDCl₃) 7.36-7.24 (m, 6H), 7.18-7.14 (m, 1H), 5.87-5.80 (d of d,1H), 5.51 (m, 2H), 4.84-4.67 (m, 3H), 4.55-4.47 (d of m, 1H), 4.26-3.94(m, 8H), 3.11-3.04 (m, 4H), 1.52 (d, 3H), 1.30 (d, 3H), 1.21 (s, 9H).

³¹P(CDCl₃) ppm, 10.74, 9.97.

MS(ESI+) m/z=610 (M+H⁺)

Example 6C:1-(6-((((Benzylamino)(2-(pivaloylthio)ethoxy)phosphoryl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-3-carbamoylpyridin-1-iumacetate salt

The crudeS-(2-(((benzylamino)((6-(3-carbamoylpyridin-1(4H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)phosphoryl)oxy)ethyl)2,2-dimethylpropanethioate(6.4 g, 10.5 mmols) was dissolved in methanol (65 ml) in a 250 ml singlenecked round bottomed flask with a magnetic stir bar. Cobalt acetatetetrahydrate (2.62 g, 10.5 mmoles) was added to the orange solution,giving a dark solution. The resulting homogeneous solution was cooled inan ice water bath for 10 minutes. Hydrogen peroxide (30% aqueoussolution) was added dropwise to the solution giving a dark green color.The cold bath was removed. After 1 hour, HPLC showed the reaction to befinished. The reaction was concentrated in vacuo, dissolved in methanol(60 ml) and again concentrated in vacuo. Addition of another portion ofmethanol, followed by 33 g of QuadraSil aminopropyl resin and stirringfor 1 hour gave a dark green color to the resin. The resin was filteredand washed with methanol (300 ml). The filtrate was concentrated invacuo to give 6.2 g of a brown foam. This material was carried onwithout further purification. HPLC showed a single main product whichwas confirmed by MS analysis.

MS (ESI+) m/z=608(M⁺)

Example 6D:1-(5-((((Benzylamino)(2-(pivaloylthio)ethoxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-3-carbamoylpyridin-1-iumTrifluoroacetate salt (Compound 6)

1-(6-((((Benzylamino)(2-(pivaloylthio)ethoxy)phosphoryl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-3-carbamoylpyridin-1-iumacetate salt (6.2 g, 9.3 mmols) was dissolved in a reagent systemconsisting of 43 ml trifluoroacetic acid, 39 ml dichloromethane and 4 mlof water. This mixture was stirred at 35° C. for 45 minutes, when HPLCindicated that the reaction was finished. The solvents were removed invacuo to give a glass, which was coevaporated with acetonitrile (3×50ml). The resulting glass was dissolved in a minimum amount ofdichloromethane and purified using 75 g of silica gel eluting withdichloromethane (300 ml), 1% MeOH/DCM (200 ml), 4% MeOH/DCM (400 ml),10% MeOH/DCM (400 ml), 20% MeOH/DCM (400 ml). The appropriate fractionswere collected to give 3.83 g of a rose colored foam.

1H NMR (D₂O) ppm: 9.30 (d, 1H), 9.03 (m, 1H), 8.89 (m, 1H), 7.42-7.17(m, 5H), 6.13 (m, 1H), 4.53 (m, 1H), 4.34 (m, 1H), 4.20-4.11 (m, 3H),4.04-3.96 (m, 4H), 3.04 (m, 2H).

³¹P(D₂O) 9.68 (bs) ppm.

MS(ES-API) m/z=568(M+)

Example 7: Benzyl((((3aR,6R,6aR)-6-(3-carbamoyl-1λ4-pyridin-1-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(p-tolyloxy)phosphoryl)alaninateTrifluoroacetate salt (Compound 7) Example 7A: Benzyl((((3aR,6R,6aR)-6-(3-carbamoylpyridin-1(4H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(p-tolyloxy)phosphoryl)alaninate

A 100 mL flask was charged with 1.75 gm (5.95 mmol)1-((3aR,4R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamideand flushed with nitrogen. The compound was dissolved in 20 mL dry DMFand treated with 11.8 mL (11.8 mmol) tert-butylmagnesium chloride (1M inTHF)) and stirred for 30 minutes. The resulting solution was treatedwith a solution 4.34 gm (11.8 mmol) benzyl(chloro(p-tolyloxy)phosphoryl) alaninate (McGuigan, et al., J. Med.Chem., 48, 3504 (2005)) in 10 mL dry DMF and the reaction wasstirred atRT. After 90 minutes the reaction was complete, with LC/MS showing alarge product peak with M+1=628. The solvent was stripped and theresidue was co-evaporated with 2×ACN, then dried in vacuo to give 9.5 gmas an amber foam. The foam was pooled with a second reaction containing3.37 mmol phosphoryl chloride starting material for purification viaflash chromatography using 5% MeOH in DCM. The pooled fractions werestripped and evaporated in vacuo to give 4 gm benzyl((((3aR,6R,6aR)-6-(3-carbamoylpyridin-1(4H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(p-tolyloxy)phosphoryl)alaninate,as a yellow oil (68%).

¹H NMR (ACN-d₃): δ 7.9 (bd s, 1H); 7.4 (m, 4H); 7.1 (m, 5H); 5.9 (m,2H); 5.13 (s, 1H); 5.10 (d, 1H); 4.8 (dd, 1H); 4.75 (m, 1H); 4.5 (m,1H); 4.15 (m, 1H); 4.0 (m, 1H); 3.0 (m, 2H); 2.3 (3, 3H); 1.5 (d, 3H);1.3 (s, 3H); 1.2 (s, 3H).

³¹P NMR (ACN-d₃): 5.1 and 5.0 ppm.

MS (ES-API⁺) m/z=628 (M+H⁺)

Example 7B:((((3aR,6R,6aR)-6-(3-carbamoyl-1λ⁴-pyridin-1-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(p-tolyloxy)phosphoryl)alaninateacetate salt

This synthesis follows the procedure described in Monatsh für Chemie,134:107 (2003). A 250 mL flask was charged with 4 gm (6.4 mmol) benzyl((((3aR,6R,6aR)-6-(3-carbamoylpyridin-1(4H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(p-tolyloxy)phosphoryl)alaninate and dissolved in 100 mL MeOH. This solution was treated with asolution of 1.89 gm (7.6 mmol) CoAc₂-4H₂O in 50 mL MeOH and then with2.75 mL 30% H₂O₂ and the dark mixture was allowed to stir at RT. After40 minutes, it was ˜95% complete by LC. The reaction was treated with anadditional 200 uL peroxide and went to completion within 30 minutes. Thereaction was treated with 14 gm Quadrapure AP silica capture resin and˜5 mL water and stirred at room temperature for ˜15 minutes, after whichthe resin was removed by filtration and washed with MeOH and water. Thefiltrate was evaporated to give 4 gm benzyl((((3aR,6R,6aR)-6-(3-carbamoyl-1λ⁴-pyridin-1-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(p-tolyloxy)phosphoryl)alaninateacetate salt as a dark oil. LC/MS showed two peaks corresponding to thediastereomers, with MS (ES-API⁺) m/z=626. This product was used directlyin the next reaction.

Example 7C: Benzyl((((3aR,6R,6aR)-6-(3-carbamoyl-1λ⁴-pyridin-1-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(p-tolyloxy)phosphoryl)alaninateTrifluoroacetate salt (Compound 7)

A 500 mL flask was charged with 4.0 gm benzyl((((3aR,6R,6aR)-6-(3-carbamoyl-1λ⁴-pyridin-1-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(p-tolyloxy)phosphoryl)alaninateacetate salt and was dissolved in 60 mL DCM. This mixture was treatedwith 60 mL of 90% TFA/H₂O and the resulting solution was stirred at 37°C. for 45 minutes. The solvent was stripped and the residue dried onhigh vacuum to give 4.32 gm as a dark oil. The oil was purified viaflash chromatography with 10%-15% MeOH in DCM, containing 1% HCO₂H and2% H₂O. The pooled product fractions were stripped and co-evaporatedwith water (3×) to remove formic acid. The product was mixed in 9:1water/ACN, frozen and lyophilized to give 2.1 gm benzyl((((3aR,6R,6aR)-6-(3-carbamoyl-1λ⁴-pyridin-1-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(p-tolyloxy)phosphoryl)alaninatetrifluoroacetate salt, as a tan solid (51%).

¹H NMR (ACN-d₃) (For many of the proton signals, the diastereomers wereclearly differentiated, as noted): δ 9.6 and 9.5 (s, 1H); 9.0 (m, 1H);8.8 (m, 1H); 8.4 (m, 1H); 8.0 (m, 1H); 7.3 (m, 5H); 7.0 (m, 4H); 6.1 and6.0 (d, 1H); 5.1 (s, 2H); 4.7 (q, 1H); 4.3-4.0 (m, 5H); 2.3 and 2.2 (s,3H); 1.4 and 1.3 (d, 3H).

³¹P NMR (ACN-d₃): 5.3 and 5.2 ppm.

MS (ES-API⁺) m/z=586 (M⁺).

Example 8:1-((2R,3R,4S)-5-(((((1-(Benzyloxy)-1-oxopropan-2-yl)amino)(naphthalen-1-yloxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-3-carbamoylpyridin-1-iumTrifluoroacetate salt (Compound 8) Example 8A: Benzyl((((3aR,6R,6aR)-6-(3-carbamoylpyridin-1(4H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(naphthalen-1-yloxy)phosphoryl)-L-alaninate

A 100 mL flask was charged with 1.4 gm (4.7 mmol)1-((3aR,4R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamideand flushed with nitrogen. The acetonide was dissolved in 20 mL dry DMFand treated with 9.2 mL (9.2 mmol) tert-butylmagnesium chloride (1M inTHF) and stirred for 30 minutes. The dark solution was treated with asolution of 3.7 gm (9.2 mmol) benzyl(chloro(naphthalen-1-yloxy)phosphoryl)-L-alaninate (Maneghesso, et al.,Antiviral Res., 94, 35 (2012)) in 5 mL dry DMF, and the reaction wasstirred at room temperature and monitored by HPLC. After 1 hour, thereaction was complete. The solvent was stripped and the residueco-evaporated with ACN (2×) and dried in vacuo to give 8.8 gm as anamber solid. The solid was purified via flash chromatography using 0-5%MeOH in DCM as the eluent, and the pooled product fractions stripped anddried on high vacuum to give 2.32 gm benzyl((((3aR,6R,6aR)-6-(3-carbamoylpyridin-1(4H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(naphthalen-1-yloxy)phosphoryl)-L-alaninateas a clear oil (74%).

¹HNMR (ACN-d₃): δ 8.2 (m, 1H); 7.9 (m, 2H); 7.7 (t, 1H); 7.6-7.2 (m,10H); 7.07 (d, 1H); 5.7 (m, 1H); 5.1 (d, 2H); 4.7 (dd, 2H); 4.4 (m,0.5H); 4.2 (m, 4H); 4.1 (d, 1H); 3.9 (m, 0.5H); 3.1 (m, 2H); 1.5 (s,3H); 1.4 (d, 3H); 1.3 (s, 3H).

³¹P NMR (ACN-d₃): −5.8 and −5.3 ppm.

MS (ES-API⁺) m/z=664 (M+).

Example 8B: Benzyl((((3aR,6R,6aR)-6-(3-carbamoyl-1λ⁴-pyridin-1-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(naphthalen-1-yloxy)phosphoryl)alaninateacetate salt

A 250 mL round-bottom flask was charged with 2.3 gm (3.5 mmol) benzyl((((3aR,6R,6aR)-6-(3-carbamoylpyridin-1(4H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(naphthalen-1-yloxy)phosphoryl)-L-alaninateand it was dissolved in 75 mL MeOH. The solution was treated with 863 mg(3.5 mmol) CoAc₂-4H₂O and stirred to dissolve. The resulting solutionwas treated with 1.15 mL 30% aqueous H₂O₂ and the mixture was stirred atRT. The reaction was monitored by HPLC. After 1 hour, the reaction hadprogressed to ˜90%. It was treated with an additional 150 μL peroxide tobring it to completion. The solution was treated with 7 gm QuadraSil APresin and 5 mL water. The mixture was stirred for 30 minutes, filteredand washed with MeOH and water. The filtrate was stripped to remove theMeOH, then frozen and lyophilized to give 2.17 gm benzyl((((3aR,6R,6aR)-6-(3-carbamoyl-1λ⁴-pyridin-1-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(naphthalen-1-yloxy)phosphoryl)alaninateacetate salt. LC/MS showed the material to be >96% purity with Thisproduct was used directly in the next reaction.

MS (ES-API⁺) m/z=662 (M+).

Example 8C:1-((2R,3R,4S)-5-(((((1-(Benzyloxy)-1-oxopropan-2-yl)amino)(naphthalen-1-yloxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-3-carbamoylpyridin-1-iumTrifluoroacetate salt (Compound 8)

A 250 mL flask was charged with 2.17 gm (2.8 mmol) benzyl((((3aR,6R,6aR)-6-(3-carbamoyl-1λ⁴-pyridin-1-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(naphthalen-1-yloxy)phosphoryl)alaninate.This was dissolved in 25 mL DCM and treated with 25 mL 90% TFA-10% H₂Oand the solution stirred at 35° C. After 3.5 hours, the solvent wasstripped and the residue co-evaporated with ACN (2×). The residue wastaken up in water and methanol, frozen and lyophilized to give 2.6 gm asa dark yellow foam. The foam was purified via flash chromatography usinga gradient of 10-20% MeOH in DCM containing 2% H₂O and 1% HCO₂H. Thepooled product fractions were stripped, co-evaporated with water (2×),frozen and lyophilized to give 1.23 gm1-((2R,3R,4S)-5-(((((1-(benzyloxy)-1-oxopropan-2-yl)amino)(naphthalen-1-yloxy)phosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-3-carbamoylpyridin-1-iumtrifluoroacetate salt (59%).

¹H NMR (ACN-d₃) (For several of the proton signals, the diastereomerswere clearly differentiated, as noted): δ 8.8-7.3 (m, 17H); 6.6 (d, 1H);6.0 and 5.9 (d, 1H); 5.2 and 5.1 (s, 2H); 4.9-4.5 (m, 5H); 1.4 and 1.2(d, 3H).

³¹P NMR (ACN-d₃): 5.5 and 5.3 ppm.

MS (ES-API⁺) m/z=622 (M+).

Example 9: 2-Ethylbutyl((((3S,4R,5R)-5-(3-carbamoyl-1λ⁴-pyridin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)alaninateTrifluoroacetate salt (Compound 9) Example 9A: 2-Ethylbutyl((((3aR,6R,6aR)-6-(3-carbamoylpyridin-1(4H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(phenoxy)phosphoryl)alaninate

A 250 mL flask was charged with 1.83 gm (6.2mmol)₁-((3aR,4R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamideand it was flushed with nitrogen. The compound was dissolved in 30 mLdry DMF and treated with 7.4 mL (7.4 mmol) tert-butylmagnesium chloride(1M in THF)) and stirred for 30 minutes. The resulting solution wastreated with a solution of 3.34 gm (7.4 mmol) 2-ethylbutyl((4-nitrophenoxy)(phenoxy)phosphoryl)alaninate (Mayes, et al., WO2013/177219) in 5 mL dry DMF and the reaction was stirred at roomtemperature and monitored by HPLC. After one hour, the reaction wascomplete and the solvent was evaporated using high vacuum. The residuewas then co-evaporated from ACN (2×) and from toluene (1×) and dried onhigh vacuum to give ˜18 gm as a bright yellow semi-solid (stillcontaining some solvent). The solid was purified via flashchromatography with a gradient of 0-5% MeOH in DCM. The pooled productfractions were stripped to give 4.4 gm 2-ethylbutyl((((3aR,6R,6aR)-6-(3-carbamoylpyridin-1(4H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(phenoxy)phosphoryl)alaninateas a clear yellow oil (106%: material contains DMF and p-nitrophenol,neither of which affect the subsequent reactions.)

¹H NMR (CDCl₃): δ 7.9 (s, 1H); 7.3-7.1 (m, 5H); 5.7 (m, 1H); 4.7-4.0(7H); 3.1 (d, 2H); 1.5 (d, 3H); 1.4-1.2 (m, 10H); 1.16 (s, 3H); 0.8 (dt,6H).

³¹P NMR (CDCl₃): 3.5 and 3.6 ppm.

MS (ES-API⁺) m/z=608 (M⁺).

Example 9B: 2-Ethylbutyl((((3aR,6R,6aR)-6-(3-carbamoyl-1λ⁴-pyridin-1-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(phenoxy)phosphoryl)alaninateacetate salt

A 250 mL flask was charged 3 gm (4.93 mmol) 2-ethylbutyl((((3aR,6R,6aR)-6-(3-carbamoylpyridin-1(4H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(phenoxy)phosphoryl)alaninatedihydroacetonide and dissolved in 100 mL MeOH. The solution was treatedwith a solution of 1.23 gm (4.93 mmol) CoAc₂-4H₂O in 25 mL MeOH. Thesolution was treated with 2 mL 30% aqueous H₂O₂ and the mixture wasallowed to stir at room temperature. The reaction was monitored by HPLCand was nearly complete in 30 minutes; an additional 100 μl peroxide wasadded to drive it to completion. After 30 minutes, the dark solution wastreated with 12 gm QuadraSil AP resin and 10 mL water. After stirringfor 15 minutes, the resin was removed by filtration and the solid washedwith MeOH and water. The filtrate was stripped to remove the MeOH andthe aqueous solution frozen and lyophilized to afford 2.54 gm2-ethylbutyl((((3aR,6R,6aR)-6-(3-carbamoyl-1λ⁴-pyridin-1-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(phenoxy)phosphoryl)alaninateacetate salt, as a dark solid, which was used directly in the nextreaction.

Example 9C: 2-Ethylbutyl((((3S,4R,5R)-5-(3-carbamoyl-1λ⁴-pyridin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)alaninateTrifluoroacetate salt (Compound 9)

A 250 mL flask was charged with 2.5 gm crude 2-ethylbutyl((((3aR,6R,6aR)-6-(3-carbamoyl-1λ⁴-pyridin-1-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(phenoxy)phosphoryl)alaninateacetate salt and it was dissolved in 40 mL DCM. The solution was treatedwith 40 mL 90% TFA/10% H₂O and the solution was stirred at 37° C. After45 minutes, the solvent was stripped and the residue evaporated on highvacuum, then co-evaporated with ACN (2×). The crude product was dilutedwith water, frozen and lyophilized to give 3.19 gm as a dark solid. Thesolid was purified via flash chromatography with a gradient of 10-15%MeOH in DCM, (containing 1% formic acid and 2% H₂O). The pooledfractions were stripped and then co-evaporated with water (3×) to removeresidual formic acid. The product was the taken up in water/ACN, frozenand lyophilized to give 1.54 2-ethylbutyl((((3S,4R,5R)-5-(3-carbamoyl-1λ⁴-pyridin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)alaninatetrifluoroacetate salt, as a tan solid (36% 2-step yield).

¹H NMR (D₂O) (For several of the proton signals, the diastereomers wereclearly differentiated, as noted): δ 9.4 and 9.3 (s, 1H); 9.2 and 9.1(d, 1H); 9.0 and 8.9 (d, 1H); 8.3 and 8.2 (t, 1H); 7.3 (m, 2H); 7.2 (m,1H); 7.1 (m, 2H); 6.3 and 6.2 (d, 1H); 4.75 (m, 1H); 4.65 (m, 1H); 4.55(m, 0.5H); 4.45 (m, 0.5H); 4.33 (m, 0.5H); 4.25 (m, 0.5H); 4.05 (m, 3H);1.5 (m, 1H); 1.4 (d, 3H); 1.3 (m, 4H).

³¹P NMR (D₂O): 5.3 and 5.1 ppm.

MS (ES-API⁺) m/z=566 (M+).

Example 10: Neopentyl((((3S,4R,5R)-5-(3-carbamoyl-1λ⁴-pyridin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)alaninateTrifluoroacetate salt (Compound 10) Example 10A: Neopentyl((((3aR,6R,6aR)-6-(3-carbamoylpyridin-1(4H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(phenoxy)phosphoryl)alaninate

A 100 mL flask was charged with 792 mg (2.7 mmol)1-((3aR,4R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamideand it was flushed with nitrogen. The compound was dissolved in 20 mLdry DMF and treated with tert-butylmagnesium chloride (1M in THF)) andstirred for 30 minutes and then treated with a solution of 1.4 gm (3.2mmol) neopentyl ((4-nitrophenoxy)(phenoxy)phosphoryl)alaninate(Menghesso, et al., Antiviral Res., 94, 35 (2012)) 5 mL dry DMF. Thereaction was stirred at room temperature overnight. The solvent wasstripped to give an oil that was co-evaporated from toluene (2×) anddried on high vacuum to give 3.1 gm. The product was pooled with productfrom a previous reaction and purified via flash chromatography using agradient of 0-10% MeOH in DCM. The pooled product fractions wereevaporated to dryness to give 2.8 gm neopentyl((((3aR,6R,6aR)-6-(3-carbamoylpyridin-1(4H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(phenoxy)phosphoryl)alaninateas an oil.

¹H NMR (CDCl₃): δ 8.0 and 7.9 (s, 2H); 7.3-7.1 (m, 5H); 6.8 and 6.7 (s,1H); 5.8 (m, 1H); 4.6 (m, 2H); 4.5 (m, 1H); 4.3 (m, 3H); 3.8 (m, 1H);3.7 (m, 1H); 3.0 (m, 2H); 1.5 (s, 3H); 1.4 (s, 3H); 1.2 d, 3H); 0.9(9H). (Contains DMF 2.9 and 2.8 ppm)

³¹P (CDCl₃): 3.6 ppm (bd).

MS (ES-API⁺) m/z=593 (M+).

Example 10B: Neopentyl((((3aR,6R,6aR)-6-(3-carbamoyl-1λ⁴-pyridin-1-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(phenoxy)phosphoryl)alaninateacetate salt

A 250 mL flask was charged with 2.7 gm (4.5 mmol) neopentyl((((3aR,6R,6aR)-6-(3-carbamoylpyridin-1(4H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(phenoxy)phosphoryl)alaninateand it was dissolved in MeOH. The solution was treated with 1.13 gm (4.5mmol) CoAc₂-4H₂O and the mixture was stirred briefly to dissolve andtreated with 2 mL 30% aqueous H₂O₂. The dark solution was stirred atroom temperature and the reaction monitored by HPLC. After 25 minutes,the reaction was complete. The solution was treated with 13 gm QuadraSilresin and 5 mL water and allowed to stir 45 minutes. The resin wasfiltered and washed with MeOH and water. The filtrate was evaporated invacuo, diluted with water, frozen and lyophilized to afford 2.25 gmneopentyl((((3aR,6R,6aR)-6-(3-carbamoyl-1λ⁴-pyridin-1-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(phenoxy)phosphoryl)alaninateacetate salt as a dark solid. This solid was used directly in the nextreaction.

MS (ES-API⁺) m/z=592 (M+).

Example 10C: Neopentyl((((3S,4R,5R)-5-(3-carbamoyl-1λ⁴-pyridin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)alaninateTrifluoroacetate salt (Compound 10)

A 250 mL flask was charged with 2.2 gm neopentyl((((3aR,6R,6aR)-6-(3-carbamoyl-1λ⁴-pyridin-1-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(phenoxy)phosphoryl)alaninate acetate salt and was dissolved in 25 mL DCM. The solution wastreated with 25 mL 90% TFA/H₂O and stirred at 37° C. After 45 minutes,the solvent was stripped on high vacuum and the residue co-evaporatedwith ACN (2×), then diluted with water, frozen and lyophilized to give3.19 gm as a dark solid. The crude product was purified via flashchromatography with a gradient of 5-15% MeOH in DCM, containing 1%HCO₂H-2% H₂O. The pooled fractions were stripped and co-evaporated withwater (3×) to remove formic acid. The product was taken up in water andACN, frozen and lyophilized to give 496 mg neopentyl((((3S,4R,5R)-5-(3-carbamoyl-1λ⁴-pyridin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)alaninatetrifluoroacetate salt as a tan solid.

¹H NMR (ACN-d₃) (For some of the proton signals, the diastereomers wereclearly differentiated, as noted): δ 9.7 and 9.6 (bd s, 1H); 9.2 and 9.0(m, 1H); 8.6 (m, 1H); 8.2 (d, 1H); 7.4-7.1 (m, 5H); 6.2 and 6.1 (m, 1H);4.85 (m, 2H); 4.6-3.9 (5H); 3.7 (m, 1H); 1.37 and 1.36 (d, 3H); 0.95(9H).

³¹P NMR (ACN-d₃): 5.4 ppm (bd)

MS (ES-API⁺) m/z=552 (M+).

Example 11:1-((2R,3R,4S)-3,4-Dihydroxy-5-(((2-oxido-4-(pyridin-3-yl)-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)tetrahydrofuran-2-yl)-1λ⁴-pyridine-3-carboxamideTrifluoroacetate salt (Compound 11) Example 11A:1-((3aR,4R,6aR)-2,2-Dimethyl-6-(((2-oxido-4-(pyridin-3-yl)-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamide

A 250 mL flask was charged with 2.0 gm (6.76 mmol)1-((3aR,4R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamideand flushed with nitrogen. The compound was dissolved in 25 mL dry DMFand treated with 10.1 mL (10.1 mmol) tert-butylmagnesium chloride (1M inTHF) and stirred for 30 minutes. The dark solution was treated with asolution of 2.5 gm (7.44 mmol)(±)-2-(4-nitrophenoxy)-4-(pyridin-3-yl)-1,3,2-dioxaphosphinane 2-oxide(Reddy, et al., J. Med. Chem., 51, 666 (2008) in 12 mL dry DMF and thereaction was stirred at room temperature overnight. The solvent wasstripped to give a dark oil which was co-evaporated from ACN (2×). Aprecipitate formed upon the addition of ACN, which was removed byfiltration and washed with ACN. The combined filtrates were evaporatedto give 1.3 gm as an oil. The oil was purified via flash chromatographywith 5-25% MeOH in DCM. The pooled product fractions were evaporated toafford 1.3 gm1-((3aR,4R,6aR)-2,2-dimethyl-6-(((2-oxido-4-(pyridin-3-yl)-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamideas a pale yellow solid that was used directly in the next reaction.

¹H NMR (DMSO-d₆): δ 8.7 (d, 1H); 8.6 (d, 1H); 7.9 (d, 1H); 7.5 (dd, 1H);6.1 (d, 1H); 4.9 (d, 1H); 4.7-3.2 (10H); 2.4 (m, 1H); 2.3 (m, 1H); 1.4(s, 3H); 1.3 (s, 3H).

³¹P NMR (DMSO-d₆): −6.1 and −6.2 ppm.

MS (ES-API⁺) m/z=493 (M⁺).

Example 11B:1-((3aR,4R,6aR)-2,2-Dimethyl-6-(((2-oxido-4-(pyridin-3-yl)-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1λ⁴-pyridine-3-carboxamideacetate salt

A flask was charged with a solution of 1.8 gm (2.5 mmol)1-((3aR,4R,6aR)-2,2-dimethyl-6-(((2-oxido-4-(pyridin-3-yl)-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamidein 50 mL MeOH. This was treated with 613 mg (2.5 mmol) CoAc₂-4H₂O andstirred to dissolve, then treated with 750 uL 30% aqueous H₂O₂. The darksolution was stirred at room temperature and the reaction monitored byLC/MS. After 45 minutes, the solution was treated with 4.5 gm QuadaSilAP resin and 5 mL water. The mixture was stirred for 90 minutes,filtered, and the resin washed with water and MeOH. The filtrate asstripped, frozen and lyophilized to afford 1.8 gm1-((3aR,4R,6aR)-2,2-dimethyl-6-(((2-oxido-4-(pyridin-3-yl)-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1λ⁴-pyridine-3-carboxamideacetate salt, as a colored solid that was used directly in the nextreaction. LC/MS shows a single product peak with MS (ES-API⁺) m/z=493(M⁺).

Example 11C:1-((2R,3R,4S)-3,4-Dihydroxy-5-(((2-oxido-4-(pyridin-3-yl)-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)tetrahydrofuran-2-yl)-1λ⁴-pyridine-3-carboxamideTrifluoroacetate salt (Compound 11)

A 250 mL flask was charged with 1.8 gm (3.3 mmol)1-((3aR,4R,6aR)-2,2-dimethyl-6-(((2-oxido-4-(pyridin-3-yl)-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1λ⁴-pyridine-3-carboxamide.This was dissolved in 25 mL DCM and treated with 25 mL 90% TFA/H₂O andthe dark solution stirred at 35° C. After 2 hours, the solvents werestripped and the residue was co-evaporated from ACN (2×). The residuewas taken up in water, frozen and lyophilized to give 3.09 gm, as a bluesolid. The solid was purified via flash chromatography with a gradientof 15-20% MeOH in DCM, containing 1% formic acid and 3% water. Theproduct fractions were pooled and stripped and co-evaporated from water(2×). The product was taken up in water, frozen and lyophilized to give620 mg1-((2R,3R,4S)-3,4-dihydroxy-5-(((2-oxido-4-(pyridin-3-yl)-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)tetrahydrofuran-2-yl)-1λ⁴-pyridine-3-carboxamidetrifluoroacetate salt, as a pale blue solid (33%).

¹H NMR (D₂O): δ 9.3 and 9.2 (s, 1H); 9.1 (m, 1H); 8.8 (m, 1H); 8.5 (m,1H); 8.1 (m, 1H); 7.9 (m, 1H); 7.5 (m, 1H); 6.1 and 6.0 (d, 1H); 4.6-4.3(9H); 2.4-2.2 (2H).

³¹P NMR (D₂O): −4.7 and −4.9 ppm.

MS (ES-API⁺) m/z=452 (M⁺).

Example 12:1-((2R,3R,4S)-5-(((4-(3-Chloro-4-fluorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-1λ⁴-pyridine-3-carboxamideTrifluoroacetate salt (Compound 12) Example 12A:1-((3aR,4R,6aR)-6-(((4-(3-Chloro-4-fluorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamide

A 500 mL round-bottomed flask was charged with 2.89 gm (9.8 mmol)1-((3aR,4R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamideand it was flushed with nitrogen. This material was dissolved in 75 mLdry DMF and treated with 14.7 mL (14.7 mmol) tert-butylmagnesiumchloride (1M in THF) and the solution was stirred for 30 minutes. Thissolution was treated with a solution of the 4.16 gm (10.7 mmol)(±)-4-(3-chloro-4-fluorophenyl)-2-(4-nitrophenoxy)-1,3,2-dioxaphosphinane2-oxide (Erion, et al. PCT Int. Appl., 2007/022073) in 75 mL dry DMF andthe reaction was warmed to 45° C. and stirred. The reaction wasmonitored by HPLC and was seen to be complete after 3 hours, whereuponit was cooled to room temperature. The reaction was stripped under highvacuum to give a thick oil. This was taken up in ˜250 mL DCM andextracted with 3× 200 mL water, then brine. The organic layer was driedover sodium sulfate, filtered and evaporated to give 5 gm as a darksolid. This was purified via flash chromatography using a gradient of0-10% MeOH in DCM. The product fractions were pooled and evaporated togive 2.65 gm1-((3aR,4R,6aR)-6-(((4-(3-chloro-4-fluorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamideas a yellow/orange solid.

¹H NMR (CDCl₃): δ 8.0 (s, ˜2H); 7.5-7.0 (m, 5H); 5.6 (d, 1H); 4.9-4.2(m, 9H); 3.1 (d, 2H); 2.3 (m, 1H); 2.1 (m, 1H); 1.5 (s, 3H); 1.3 (s,3H). Residual DMF also present.

³¹P NMR (CDCl₃): −4.8 and −5.0 ppm.

MS (ES-API⁺) m/z=545 (M+).

Example 12B:1-((3aR,4R,6aR)-6-(((4-(3-chloro-4-fluorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1λ⁴-pyridine-3-carboxamideacetate salt

A 250 mL flask was charged with 2.15 gm (3.95 mmol)1-((3aR,4R,6aR)-6-(((4-(3-chloro-4-fluorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamideand treated with a solution of 982 mg (3.95 mmol) CoAc₂-4H₂O in 100 mLMeOH that had been cooled to 0° C. The solution was treated with 900 μL30% H₂O₂, and the mixture was allowed to warm to room temperature andthe reaction monitored by HPLC. The reaction was ˜50% complete after 1hour and was treated with an additional 950 μL H₂O₂ over the next threehours. The reaction was then treated with 10 gm Quadrasil AP and 10 mLwater and stirred at room temperature for 90 minutes. The resin wasremoved by filtration, the solid washed with water and MeOH and thefiltrate stripped to afford 3 gm1-((3aR,4R,6aR)-6-(((4-(3-chloro-4-fluorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1λ⁴-pyridine-3-carboxamideacetate salt as a dark solid. The crude product was used directly innext reaction.

MS (ES-API⁺) m/z=543 (M+)

Example 12C:1-((2R,3R,4S)-5-(((4-(3-Chloro-4-fluorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-1λ⁴-pyridine-3-carboxamideTrifluoroacetate salt (Compound 12)

A 500 mL flask was charged with 2.4 gm (3.92 mmol)1-((3aR,4R,6aR)-6-(((4-(3-chloro-4-fluorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1λ⁴-pyridine-3-carboxamideacetate salt. This compound was dissolved 25 mL DCM and treated with 25mL 90% TFA/H₂O. The dark solution was stirred at 35° C. for 1 hour. Thesolvent was stripped on high-vacuum and co-evaporated with ACN (2×). Theresidue was diluted with water, frozen, and lyophilized to give 3 gm ofa dark semi-solid. This was purified via flash chromatography using agradient of 10-30% MeOH in DCM, containing 1% formic acid and 2% water.The pooled product fractions were pooled, stripped, co-evaporated fromwater, taken up in water, frozen and lyophilized to give 510 mg1-((2R,3R,4S)-5-(((4-(3-chloro-4-fluorophenyl)-2-oxido-1,3,2-dioxaphosphinan-2-yl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-1λ⁴-pyridine-3-carboxamidetrifluoroacetate salt, as a pale yellow solid (21%). LC/MS shows twopeaks corresponding to the two diastereomers, each with the same mass.MS (ES-API⁺) m/z=503 (M+).

¹H NMR (D₂O) (For several of the proton signals, the diastereomers wereclearly differentiated, as noted): δ 9.3 and 9.2 (s, 1H); 9.1 and 9.0(d, 1H); 8.6 (m, 1H); 8.1 (m, 1H); 7.3 (m, 3H); 6.2 and 6.1 (d, 1H);4.5-3.6 (m, 10H); 2.1 (m, 1H); 1.9 (m, 1H).

³¹P NMR (D₂O): 4.5 and 4.9 ppm; 5.9 and 6.3 ppm. These signalscorrespond to the four diastereomers generated from chiral centersproduced at the racemic diol and the phosphorus.

Example 13:((2R,3S,4R,5R)-5-(3-Carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methylisopropyl phosphate (Compound 13)

A 50 mL receiving flask was charged with 2.15 g (5.32 mmol) of3-carbamoyl-1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyridin-1-iumtrifluoromethanesulfonate (nicotinamide ribosidetrifluoromethanesulfonate salt). (Sauve et al. WO2007061798) Thismaterial was suspended in 12 mL of anhydrous acetonitrile, thenconcentrated in vacuo (35° C., ca. 1 torr) to a foam. A stir bar wasadded, and the flask was placed under argon. To this was added 13 mL oftrimethylphosphate. The mixture was cooled with an ice bath, then 973microliters (10.6 mmol) of phosphorus oxychloride was added via syringe,in one portion. The reaction was stirred while cooling with an ice bathfor 6.75 h, then 3.5 g (58.2 mmol) of isopropanol was added in oneportion. This was allowed to stand at 4° C. for 24 h, then 5 mL of waterwas added in one portion, and the mixture was allowed to stand at 4° C.for an additional 15 h.

Immediately prior to chromatography, the reaction mixture was dilutedwith 30 mL of ethyl acetate. A 100 g Quadrasil AP column was conditionedaccording to the procedure described in J. Org. Chem., 2012, 77,7319-7329. Following conditioning, the column was pre-equilibrated withethyl acetate, and topped with 10 mL of ethyl acetate. The crudereaction mixture in ethyl acetate was added to the top of the column,then eluted onto the column. The column was eluted sequentially with 200mL of ethyl acetate, then with 550 mL of 60:40 (v:v) ethyl acetate:methanol, collecting 35 mL fractions. Fractions were monitored by LCMSfor the presence of product (m/z=377 (M+H)⁺). Fractions that were >98%pure by LCMS were pooled and concentrated in vacuo to a white foam. Anadditional 35 mL of 60:40 ethyl acetate: methanol was added to the foam,and crystals formed. The crystals were filtered, and dried in vacuo togive 197 mg (10%) of the title product. The supernatant was concentratedin vacuo. This concentration residue was stirred with 80:20 ethylacetate: methanol to give additional crystals. These crystals werefiltered, washed with 90:10 ethyl acetate: methanol, then dried in vacuoto give 329 mg (17%) of the title product. The total yield was 526 mg(27%) of a colorless, crystalline solid.

¹H NMR (500 MHz, D₂O) δ 9.42 (d, 1H, J=1.5 Hz), 9.24 (d, 1H, J=6.3 Hz),8.95 (dt, 1H, J=8.1, 1.5 Hz), 8.26 (dd, 1H, J=8.1, 6.3 Hz), 6.18 (d, 1H,J=5.5 Hz), 4.59 (quintet, 1H, J=2.5 Hz), 4.50 (t, 1H, J=5.2 Hz), 4.39(dd, 1H, J=5.0, 3.0 Hz), 4.38-4.32 (m, 1H), 4.26 (ddd, 1H, J=12.0, 4.5,2.4 Hz), 4.10 (ddd, 1H, J=12.0, 5.1, 2.2 Hz), 1.19 (dd, 6H, J=6.2, 2.3Hz).

MS (ES-API⁺) m/z=377.0 (M+H⁺).

Example 14:((2R,3S,4R,5R)-5-(3-Carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methylethyl phosphate (Compound 14)

This compound was prepared according to Example 13, substituting ethylalcohol for isopropanol. The product was purified in a similar way,eluting over a 100 g Quadrasil AP column first with ethyl acetate toremove the reaction solvent, then with 50:50 ethyl acetate: methanol toremove impurities, and finally with 30:70 ethyl acetate: methanol toisolate the product. Concentration of the product containing fractionsfollowed by dissolution of the residue in water and lyophilization gave293 mg (15%) of the product as a white solid.

¹H NMR (500 MHz, D₂O) δ 9.41 (d, 1H, J=1.6 Hz), 9.23 (dd, 1H, J=6.3, 1.2Hz), 8.95 (dt, 1H, J=8.1, 1.6 Hz), 8.26 (dd, 1H, J=8.1, 6.3 Hz), 6.18(d, 1H, J=5.4 Hz), 4.60 (quintet, 1H, J=2.6 Hz), 4.50 (t, 1H, J=5.2 Hz),4.39 (dd, J=5.1, 2.7 Hz), 4.27 (ddd, 1H, J=12.0, 4.4, 2.4 Hz), 4.11(ddd, 1H, J=12.0, 5.2, 2.3 Hz), 3.87 (quintet, 2H, J=7.2 Hz), 1.19 (td,3H, J=7.1, 0.5 Hz).

MS (ES-API⁺) m/z=362.9 (M+H⁺).

Example 15:((2R,3S,4R,5R)-5-(3-Carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methylpropyl phosphate (Compound 15)

This compound was prepared according to Example 13, substitutingn-propyl alcohol for isopropanol and stirring for 1.25 h instead of 24 hbefore the addition of water. The product was purified in a similar way,eluting over a 100 g Quadrasil AP column first with ethyl acetate toremove the reaction solvent, then with 70:30 ethyl acetate: methanol toelute the product. The product from the column was recrystallized from70:30 ethyl acetate: methanol to give 771 mg (40%) of the product as acolorless crystalline solid.

¹H NMR (500 MHz, D₂O) δ 9.42 (m, 1H), 9.23 (m, 1H), 8.95 (dt, 1H, J=8.1,1.5 Hz), 8.26 (dd, 1H, J=8.1, 6.4 Hz), 6.18 (d, 1H, J=5.4 Hz), 4.60(quintet, 1H, J=2.5 Hz), 4.50 (t, 1H, J=5.2 Hz), 4.40 (dd, 1H, J=5.1,2.7 Hz), 4.27 (ddd, J=12.0, 4.4, 2.4 Hz), 4.11 (ddd, 1H, J=12.0, 5.2,2.2 Hz), 3.76 (m, 2H), 1.56 (m, 2H), 0.84 (t, 3H, J=7.4 Hz).

MS (ES-API⁺) m/z=377.0 (M+H⁺).

Example 16:((2R,3S,4R,5R)-5-(3-Carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methylphosphate (Compound 16)

A 100 mL receiving flask was charged with 4.62 g (11 mmol) of3-carbamoyl-1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyridin-1-iumtrifluoromethanesulfonate (nicotinamide ribosidetrifluoromethanesulfonate salt). To this was added 50 mL of anhydrousacetonitrile, then the mixture was stirred at ambient temperature for 15min, and concentrated in vacuo to a light yellow foam. The foam wasmaintained under an argon atmosphere. To the foam was added 25 mL oftrimethylphosphate via syringe at ambient temperature. The mixture wasstirred at ambient temperature for 5 min, then cooled with an ice bath.To this was added 4.2 mL (45.9 mmol) of POCl₃. The mixture was stirredwith ice cooling for 3 h, then the solution of3-carbamoyl-1-((2R,3R,4S,5R)-5-(((dichlorophosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)pyridin-1-iumchloride/trifluoromethanesulfonate was used in subsequent phosphateester preparations.

A 9.5 g quantity of the above reaction mixture was placed into a 50 mLreceiving flask under argon and cooled with an ice bath. To this wasadded 1.0 mL of methanol, then the mixture was stirred for 10 min. Next,6 mL of water was added, then the solution was stored at 4° C. for threedays. The product was purified by eluting over a 100 g Quadrasil APcolumn which had been conditioned according to the procedure describedin J. Org. Chem. 2012, 77, 7319-7329, and subsequently placed in ethylacetate. The reaction mixture was diluted with 60 mL of ethyl acetateand 10 mL of methanol. This was eluted onto the column, then the columnwas eluted with 100 mL of 80:20 (v:v) ethyl acetate: methanol, followedby 450 mL of 30:70 ethyl acetate: methanol. Approximately 20 mLfractions were collected during the 30:70 ethyl acetate: methanolelution. Product containing fractions were identified by LCMS. Thesewere pooled and concentrated in vacuo to an oily residue. This wasfollowed by 2×3 mL with water to remove the residual organic solvents.The residue was taken up in 3 mL of water and filtered through a cottonplug, then the cotton plug was rinsed with an additional 7 mL of water.The aqueous solution was frozen and lyophilized to give 263 mg (18%) ofa colorless solid. The product purity was >98% by LCMS, monitoring at214 nm.

¹H NMR (500 MHz, D₂O) δ 9.41 (m, 1H), 9.22 (m, 1H), 8.95 (dt, 1H, J=8.1,1.5 Hz), 8.26 (dd, 1H, J=8.0, 6.4 Hz), 6.18 (d, 1H, J=5.4 Hz), 4.60(quintet, 1H, J=2.6 Hz), 4.50 (t, 1H, J=5.2 Hz), 4.40 (dd, 1H, J=5.1,2.8 Hz), 4.27 (ddd, 1H, J=12.0, 4.4, 2.4 Hz), 4.11 (ddd, 1H, J=12.0,5.2, 2.3 Hz), 3.52 (d, 3H, J=10.8 Hz).

MS (ES-API⁺) m/z=349.0 (M+H⁺).

Example 17:3-Carbamoyl-1-((2R,3R,4S,5R)-5-(((dimethoxyphosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)pyridin-1-iumAcetate salt (Compound 17)

A 100 mL receiving flask was charged with 4.62 g (11 mmol) of3-carbamoyl-1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyridin-1-iumtrifluoromethanesulfonate (nicotinamide ribosidetrifluoromethanesulfonate salt). To this was added 50 mL of anhydrousacetonitrile, then the mixture was stirred at ambient temperature for 15min, and concentrated in vacuo to a light yellow foam. The foam wasmaintained under an argon atmosphere. To the foam was added 25 mL oftrimethylphosphate, via syringe at ambient temperature. The mixture wasstirred at ambient temperature for 5 min, then cooled with an ice bath.To this was added 4.2 mL (45.9 mmol) of POCl₃. The mixture was stirredwith ice cooling for 3 h, then the solution of3-carbamoyl-1-((2R,3R,4S,5R)-5-(((dichlorophosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)pyridin-1-iumchloride/trifluoromethanesulfonate was used in subsequent phosphateester preparations. A 10 g quantity of this solution was added to 10 mLof ice-cold methanol. The solution was stirred with ice cooling for 45min.

The product was purified by eluting over a 100 g Quadrasil AP columnwhich had been conditioned according to the procedure described in J.Org. Chem. 2012, 77, 7319-7329, and subsequently placed in ethylacetate. The reaction mixture was diluted with 90 mL of ethyl acetateand eluted onto the column. The column was then eluted with 250 mL ofethyl acetate, followed by 400 mL of 70:30 ethyl acetate: methanol, andcollecting 20 mL fractions during the ethyl acetate: methanol elution.The product containing fractions were identified by LCMS. The productcontaining fractions were pooled and concentrated in vacuo to an oilyresidue. This was followed by 3 mL of water, followed by 3 mL ofmethanol to give the product as a hygroscopic amorphous solid, 133 mg(8%).

¹H NMR (500 MHz, D₂O) δ 9.38 (m, 1H), 9.15 (m, 1H), 8.95 (dt, 1H, J=8.1,1.5 Hz), 8.26 (dd, 1H, J=8.1, 6.3 Hz), 6.23 (d, 1H, J=4.6 Hz), 4.62 (dt,1H, J=6.5, 3.2 Hz), 4.55 (ddd, 1H, J=12.0, 4.9, 2.5 Hz), 4.44 (t, 1H,J=4.8 Hz), 4.40 (ddd, 1H, J=12.0, 5.6, 3.1 Hz), 4.36 (dd, 1H, J=5.0, 4.1Hz), 3.77 (d, 3H, J=5.3 Hz), 3.75 (d, 3H, J=5.3 Hz), 1.87 (s, 3H).

MS (ES-API⁺) m/z=363.0 (M⁺).

Example 18:3-Carbamoyl-1-((2R,3R,4S,5R)-5-(((diisopropoxyphosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)pyridin-1-iumAcetate salt (Compound 18)

A 100 mL receiving flask was charged with 4.62 g (11 mmol) of3-carbamoyl-1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyridin-1-iumtrifluoromethanesulfonate (nicotinamide ribosidetrifluoromethanesulfonate salt). To this was added 50 mL of anhydrousacetonitrile, then the mixture was stirred at ambient temperature for 15min, and concentrated in vacuo to a light yellow foam. The foam wasmaintained under an argon atmosphere. To the foam was added 25 mL oftrimethylphosphate via syringe at ambient temperature. The mixture wasstirred at ambient temperature for 5 min, then cooled with an ice bath.To this was added 4.2 mL (45.9 mmol) of POCl₃. The mixture was stirredwith ice cooling for 3 h, then the solution of3-carbamoyl-1-((2R,3R,4S,5R)-5-(((dichlorophosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)pyridin-1-iumchloride/trifluoromethanesulfonate was used in subsequent phosphateester preparations. An aliquot of this solution containing approximately1.15 g of the dichlorophosphoryl intermediate was diluted with 7 mL ofisopropanol, then the mixture was stirred at ambient temperature for twodays.

The product was purified by eluting over a 100 g Quadrasil AP columnusing a sequence similar to that used to purify3-carbamoyl-1-((2R,3R,4S,5R)-5-(((dimethoxyphosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)pyridin-1-iumacetate. After concentration of the product containing fractions, theresidue was chased 2×5 mL water, avoiding concentrating to dryness andconcentrating to about 2 mL residual volume each time. The residualsolution was diluted with 5 mL of water, filtered through a 0.45 micronfilter, then the filter was washed 2×3 mL water. The combined filtrateand washings were frozen and lyophilized to give 413 mg (28%) of anamorphous solid. ¹H NMR (500 MHz, D₂O) δ 9.38 (m, 1H), 9.16 (m, 1H),8.97 (dt, 1H, J=8.1, 1.5 Hz), 8.27 (m, 1H), 6.23 (d, 1H, J=4.7 Hz), 4.63(m, 3H), 4.49 (ddd, 1H, J=12.0, 4.7, 2.5 Hz), 4.42 (t, 1H, J=4.9 Hz),4.34 (m, 2H), 1.89 (s, 3H), 1.27 (m, 9H), 1.23 (d, 3H, J=6.2 Hz). MS(ES-API⁺) m/z=419.0 (M⁺).

Example 19:3-Carbamoyl-1-(5-(((diethoxyphosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)pyridin-1-ium Chloride (Compound 19)

Nicotinamide Riboside Triflate salt (1.3 g, 3.22 mmoles) was placed intoa 10 ml single necked round bottomed flask under N₂. Freshly distilledtrimethyl phosphate (2.6 ml) was added via syringe and this was stirreduntil a solution formed (15 min). This solution was placed under vacuumfor 15 minutes to remove volatiles then placed under N₂. The solutionwas cooled in an ice-water bath for 10 minutes then phosphorousoxychloride (1.30 ml, 2.14 g, 13.8 mmols, 4.3 equivalents) was addeddropwise over 10 minutes. The reaction was stirred at 0° C. for 1 hour,placed into a 4° C. refrigerator and allowed to react overnight. Thereaction was checked by HPLC for completeness and then placed into anice-water bath and ethanol (5.0 ml, 3.95 gm, 85.6 mmoles, 26.6equivalents) was added dropwise over 6 minutes. The reaction was removedfrom the cold bath and it was allowed to warm to room temperature for 4hours. At this time, the reaction was added dropwise to a well stirredsolution of diethyl ether (100 ml). The stirring was stopped and theupper layer decanted off. The heavy oil was dissolved in a minimum ofethanol and added dropwise to well-stirred diethyl ether. The stirringwas stopped and the ether layer decanted. The resulting heavy oil wasplaced under vacuum and the resulting foam was purified using a silicagel preparative chromatography plate using 7:3:0.5 DCM:MeOH:formic acid.This gave 0.760 g of a clear oil, which still contained a small amountof trimethyl phosphate, but which was identified as diethyl NMN byHPLC/MS, ¹H and ³¹P NMR.

¹HNMR (D₂O) δ 9.31 (bs, 1H), 9.08 (m, 1H), 8.88 (1H, d), 8.21 (m, 2H),6.15 (1H, d), 4.55-4.45 (2H, m), 4.34-4.25 (3H, m), 4.04 (4H, m),1.22-1.13 (6H, m)

³¹P NMR (D₂O) δ −2.21

MS(ESI+) m/z=391(M+)

Example 20:3-Carbamoyl-1-(5-(((dibutoxyphosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)pyridin-1-ium Chloride (Compound 20)

Nicotinamide riboside triflate salt (1.57 g, 3.90 mmols) was placed intoa 10 ml single necked round bottomed flask and co evaporated with dryACN (2 ml). Freshly distilled trimethyl phosphate (2.0 ml) was added viasyringe and this was stirred until homogeneous (approximately 30minutes). The solution was degassed for 5 minutes then placed under N₂.It was cooled in an ice water bath for 10 minutes then phosphorousoxychloride (0.73 ml, 1.20 g, 7.8 mmols, 2 equiv.) was added dropwisevia syringe over 5 minutes. The reaction was kept in the cold bath for30 minutes then placed in a 4° C. refrigerator overnight. The reactionwas monitored by an H₂O quench followed by HPLC, once complete, thereaction was placed into an ice water bath and n-BuOH (2 ml, 1.62 g,21.9 mmoles, 5.6 equiv.) was added dropwise via syringe. It was stirredfor 30 minutes then placed into the refrigerator for 2 days. At thistime HPLC analysis showed approximately 60% of the diester had formed sodiethyl ether was added and the resulting clear heavy oil was placedunder vacuum. Isolation of the product was accomplished by preparativesilica gel separation using a 7:3:0.5 DCM:MeOH:formic acid system.

¹H NMR (D₂O) δ ppm 9.37 (1H, bs), 9.13 (1H, d), 8.95 (1H, d), 8.23 (1H,m), 6.18 (1H, d), 4.56-4.28 (5H, m), 3.99 (4H, q), 1.51 (4H, q),1.31-1.18 (4H, m), 0.78 (6H, t).

³¹P (D₂O) δ −0.27.

MS(ESI+) m/z=447.10 (M+)

Example 21:3-Carbamoyl-1-(3,4-dihydroxy-5-(((methoxy(3-(pentadecyloxy)propoxy)phosphoryl)oxy)methyl)tetrahydrofuran-2-yl)pyridin-1-iumTrifluoroacetate salt (Compound 21) Example 21A:3-(pentadecyloxy)propan-1-ol

This intermediate was prepared according to Yamano et al., Bioorg & Med.Chem. 20, 3658 2012.

¹H NMR (CDCl₃) δ ppm 3.77 (2H, t), 3.61 (2H, t), 3.42 (2H, t), 1.83 (2H,t), 1.562H, m), 1.31-1.25 (24H, m), 0.88 (3H, t).

Example 21B: 3-(nonyloxy)propan-1-ol

This intermediate was prepared according to Yamano et al., Bioorg & Med.Chem. 20, 3658 2012.

¹HNMR (CDCl₃) δ 3.78 (2H, t), 3.61 (2H, t), 3.43 (2H, t), 1.83 (2H, dt),1.57 (2H, dd), 1.33-1.25 (12H, m), 0.88 (3H, t).

Example 21C: Methyl (4-nitrophenyl)(3-(pentadecyloxy)propyl) phosphate

Dichloro-4-nitrophenylphosphate (1.0 g, 3.91 mmols) was placed into adry 25 ml single necked round bottomed flask equipped with a septumunder N₂. Dry THF (6 ml) was added via syringe. This solution wasstirred and cooled to −78° C. Triethylamine (1.64 ml, 1.19 g, 11.7mmols) was added dropwise to the stirred reaction mixture, giving ayellow color to the reaction. The C-15 ether (1.12 g, 3.91 mmoles),dissolved in dry THF (3 ml), was added dropwise to the reaction mixtureover 5 minutes. The cold bath was then removed and the reaction allowedto slowly warm to room temperature. A thick precipitate of triethylaminehydrochloride formed. After 40 minutes at room temperature, the reactionwas cooled with an ice bath, and dry methanol (0.158 ml, 0.125 g, 3.91mmols) was added dropwise over 2 minutes. The cold bath was then removedand the reaction allowed to warm to room temperature for 1 hour. Thereaction was then concentrated in vacuo, dissolved in ethyl acetate,filtered, re-concentrated, dissolved in hexanes, re-filtered andconcentrated in vacuo to give a thick oil, 1.95 g. This was dissolved ina minimum of dichloromethane and purified using 20 g silica gel, elutingwith 5:1 dichloromethane:ethyl acetate. This gave 1.54 g of the titleintermediate (78.6% yield).

¹H NMR (CDCl₃) δ ppm 8.24-8.21 (2H, m), 7.38-7.35 (2H, m), 4.30-4.26(2H, m), 3.90-3.86 (3H, m), 3.47 (2H, t), 3.36 (2H, t), 1.95 (2H, td),1.51 (2H, t), 1.27-1.23 (24H, m), 0.85 (3H, t).

³¹P NMR (CDCl₃) δ −5.38

Example 21D: Methyl (4-nitrophenyl)(3-(nonyloxy)propyl) phosphate

The title intermediate was prepared using the Example 23C procedureutilizing 6.33 g (24.7 mmols) of the dicholoro 4-nitrophenylphosphate,7.94 ml (5.75 g, 56.8 mmols) of triethylamine, 5.00 g (24.7 mmols) of3-(nonyloxy)propan-1-ol and 1.00 ml (0.79 g, 24.7 mmols) of anhydrousmethanol. After purification using 70 g of silica gel and 2:1hexanes:ethyl acetate as eluent, 5.00 g of the title intermediate wasobtained (48.5%).

¹H NMR (CDCl₃) δ ppm 8.27-8.25 (2H, m), 7.41-7.39 (2H, m), 4.33-4.29(2H, m), 3.91 (3H, dd), 3.50 (2H, t), 3.39 (2H, m), 2.00-1.97 (2H, m),1.55 (2H, t), 1.33-1.27 (12H, m), 0.89 (3H, t).

MS(ES-API) m/z=418(M+H+)

Example 21E:(6-(3-Carbamoylpyridin-1(4H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methylmethyl (3-(pentadecyloxy)propyl) phosphate

The title intermediate was prepared according to the procedure ofExample 6B. Using1-(6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamide(0.759 g, 2.53 mmols), 2.93 ml of 1M t-Butyl magnesium chloride in THFand 1.33 g (2.66 mmols) of methyl (4-nitrophenyl)(3-(pentadecyloxy)propyl) phosphate, the desired crude product wasobtained (2.2 g). This was purified using 20 g of silica gel and astepwise gradient of 0-10% methanol in dichloromethane as eluent. Thisgave 699 mg of product (40% yield).

¹H NMR (CDCl₃) δ ppm 7.05 (1H, s), 5.88-5.86 (1H, m), 5.61-5.58 (1H, m),4.84-4.78 (1H, m), 4.68 (1H, ddd), 4.61-4.58 (1H, m), 4.22-4.08 (4H, m),3.78-3.73 (3H, m), 3.48-3.35 (4H, m), 3.09-3.08 (1H, m), 1.93-1.89 (2H,m), 1.55-1.49 (5H, m), 1.34-1.19 (27H, m), 0.86 (3H, t).

³¹P NMR(CDCl₃) δ −0.36.

MS(ES-API) m/z=659.3 (M+H+)

Example 21F:6-(3-Carbamoylpyridin-1(4H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methylmethyl (3-(nonyloxy)propyl) phosphate

The title intermediate was prepared according to the procedure ofExample 6B. Using 3.39 g (11.4 mmols) of1-(6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-1,4-dihydropyridine-3-carboxamide,12.5 ml of 1M t-butyl magnesium chloride and 5.00 g (11.98 mmols) ofmethyl (4-nitrophenyl) (3-(nonyloxy)propyl) phosphate, the crude productwas obtained (10.62 g). This was purified using 100 g of silica gel anda stepwise gradient of 0 to 10% methanol in dichloromethane resulting inthe isolation of 2.35 g the product.

¹H NMR (CDCl₃) δ ppm 7.10 (1H, s), 5.914 (1H, d), 5.59-5.581H, m),4.88-4.82 (2H, m), 4.73 (1H, m), 4.63 (1H, m), 4.26-4.12 (5H, m),3.82-3.78 (3H, m), 3.52-3.49 (2H, m), 3.41 (2H, m), 1.98-1.93 (2H, m),1.58-1.54 (5H, m), 1.39-1.28 (15H, m), 0.90 (3H, t).

MS (ES-API) m/z=575(M+H+)

Example 21 G:3-Carbamoyl-1-(6-(((methoxy(3-(pentadecyloxy)propoxy)phosphoryl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pyridin-1-iumacetate

The title intermediate was prepared according to the procedure ofExample 6C. Using 2.07 g (3.14 mmoles) of(6-(3-carbamoylpyridin-1(4H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methylmethyl (3-(pentadecyloxy)propyl) phosphate, the starting material wasoxidized and purified to give 1.18 g of product (52% yield).

¹H NMR (CDCl₃) δ ppm 9.86 (1H, d), 9.18-9.13 (2H, m), 8.19 (1H, s),6.45-6.43 (1H, m), 6.24 (1H, d), 5.17-5.16 (1H, m), 4.91 (1H, d), 4.85(1H, dt), 4.12 (2H, dd), 3.72 (3H, dd), 3.45 (2H, m), 3.38 (2H, m), 1.90(3H, m), 1.65 (3H, bs), 1.55 (2H, m), 1.39 (3H, bs), 1.25 (24H, bs),0.88 (3H, t).

MS(ES-API) m/z=657.5 (M+)

Example 21H:3-Carbamoyl-1-(6-(((methoxy(3-(nonyloxy)propoxy)phosphoryl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pyridin-1-iumacetate

The title intermediate was prepared according to the procedure ofExample 6C. Using 2.40 g, (4.17 mmoles) of6-(3-carbamoylpyridin-1(4H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methylmethyl (3-(nonyloxy)propyl) phosphate, the starting material wasoxidized to give 2.05 g of a green foam (78% yield). The purity wasassessed by HPLC to be sufficient to carry on to the final product, theMS confirmed its identity.

MS(ES_API) m/z=573(M+)

Example 21I:3-carbamoyl-1-(3,4-dihydroxy-5-(((methoxy(3-(pentadecyloxy)propoxy)phosphoryl)oxy)methyl)tetrahydrofurantetrahydrofurantetrahydrofuran-2-yl)pyridin-1-iumTrifluoroacetate salt (Compound 21)

Compound 21 was prepared according to the procedure of Example 6D. 2.90g (4.05 mmoles) of3-carbamoyl-1-(6-(((methoxy(3-(pentadecyloxy)propoxy)phosphoryl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pyridin-1-iumacetate was treated with triflic acid, dichloromethane and water(22:20:1.9 ml) at 35° C. for 2 hours. When the reaction was complete byHPLC, the reaction was concentrated in vacuo and co-evaporated withacetonitrile (2×15 ml), giving a dark green glass. This was dissolved ina minimum of dichloromethane and loaded onto a 50 g silica gel columnand eluted with a step-wise gradient of 0 to 20% methanol indichloromethane. Concentration of the product fractions gave 1.74 g (56%yield).

¹HNMR (CDCl₃) δ ppm 9.93 ((1H, bs), 9.42 (1H, bs), 9.09 (1H, bd), 9.00(1H, bs), 8.16 (1H, m), 6.55 (1H, bs), 6.35 (bs), 4.64 (1H, bs), 4.48(2H, bs), 4.37 (2H, bs), 4.23-4.14 (2H, m), 3.78 (3H, dd), 3.51 (2H,dt), 3.41 (2H, td), 1.95 (2H, td), 1.56 (2H, t), 1.32 (25H, m), 0.905(3H, t).

³¹P NMR (CDCl₃) δ 0.26 (bs).

MS(ES-API) m/z=617(M+)

Example 22:3-Carbamoyl-1-(3,4-dihydroxy-5-(((methoxy(3-(nonyloxy)propoxy)phosphoryl)oxy)methyl)tetrahydrofuran-2-yl)pyridin-1-ium Trifluoroacetate salt(Compound 22)

Compound 22 was prepared according to the procedure of Example 6D.3-carbamoyl-1-(6-(((methoxy(3-(nonyloxy)propoxy)phosphoryl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pyridin-1-iumacetate (2.05 g, 3.57 mmoles) was treated with trifluoroacetic acid,dichloromethane and water (14:15.5:1.34 ml) at 35° C. for 1.5 hours. Thereaction was monitored by HPLC and when finished, it was concentrated invacuo and co-evaporated 3 times with acetonitrile (15 ml each). Thisgave a dark green oil, 3.0 g. The oil was dissolved in a minimum ofdichloromethane and purified on 30 g of silica gel utilizing a step-wisegradient of 0 to 20% methanol in dichloromethane. The product fractionswere collected and concentrated to give 1.08 g of a brown glass, whichwas dissolved in 3 ml of water, frozen and lyophilized to give 1.00 g(41% yield) of a light brown foam.

¹H NMR (D₂O) δ ppm 9.39 (1H, bs), 9.17-9.15 (1H, m), 8.98 (1H, dd), 8.27(1H, t), 6.24-6.23 (1H, m), 4.58-4.4.57 (1H, m), 4.51-4.46 (1H, m),4.42-4.36 (2H, m), 4.32-4.29 (1H, m), 4.13 (2H, q), 3.74 (3H, dd),3.49-3.45 (2H m), 3.39-3.30 (2H, m), 1.89 (2H, td), 1.48-1.47 (2H, m),1.21 (12H, bs), 0.80 (3H, t).

³¹P NMR (D₂O) δ 0.15 (d).

MS(ES-API) m/z=533(M+)

Example 23: Biological Assays for Determining Levels of NAD+

As the compounds disclosed herein can act as prodrugs for NMN, theirbiological activity was assessed in vivo by measuring the ability toelevate NAD+ levels in liver and skeletal muscle. These tissues wereselected for pharmacodynamics studies due to their relevance for thetreatment of metabolic diseases. In general, C57/BL6 mice were fastedfrom 16 hours, then administered a 500 mg/ml oral dose of a disclosedcompound in PBS buffer by oral gavage. In some cases, the dose was 250mg/kg or in an alternate vehicle: ethanol/PBS/PEG400 (10/30/60). Afterdosing, the mice were sacrificed at pre-set time points after dosing(generally 4, 8 and 24 hours). The liver and skeletal tissues wereremoved and flash frozen. After some period of frozen storage, tissuehomogenates were prepared. The level of NAD+ was quantified using LC/MSmethods.

For bioanalysis, a method of standard addition was used to quantify theNAD. Each homogenate was divided and certain aliquots had knownconcentrations of exogenous NAD added. The NAD present in the originalsample was then calculated by linear regression. Data are presented as aratio of mean NAD concentration in treated mouse tissues to mean NADconcentration in the vehicle control group tissues at the paired timepoints.

Example 23A: Study Design for Assaying Levels of NAD+ in Mice

Mixtures of compound and PBS, pH 7 were formulated ˜1 hour before dosingand remained stirring until dose completion. Residual formulated testmaterials were stored at −20±5° C. until discarded. Naïve male C57/BL6mice at ˜25 g at dose initiation, age as appropriate for weight wereassigned to dose groups of six animals per dose. Dosing treatments wereadministered via a single oral gavage dose (PO) The dose volume for eachanimal (5 mL/kg) was based on the most recent body measurements takenthe morning of dosing. Animals were fasted at least 16 hrs prior todose, with food returned at least 4 hours post dose.

Liver and skeletal muscle were collected and analyzed for nicotinamideadenine dinucleotide (NAD) at 0, 4, 8, and 24 hours post-dose. For eachmouse, the entire liver was removed, rinsed with saline, blotted dry andfrozen at −70° C. to store for NAD concentration analysis. Also, thesoleus skeletal muscle was removed from the gastrocnemius, rinsed withsaline, blotted dry and frozen at −70° C. to store for NAD concentrationanalysis.

After thawing samples for analysis, the tissues were homogenized andextracted as follows:

Tissue Homogenization Procedure

-   -   1. Add 5× (tissue wt.*5) mL 0.1M ZnSO₄ into all tubes.    -   2. Add 5× (tissue wt*5) mL Methanol into all tubes.    -   3. Homogenize each sample until completely liquid.    -   4. Vortex each sample.        Tissue Extraction Procedure    -   1. On ice, aliquot 20 μL of standards, control blanks, and        matrix blanks into a 96-well plate.    -   2. Aliquot 20 μL of sample into sample wells.    -   3. Add 100 μL of IS in 80:20 Methanol:water into each well,        except blanks add 100 μL of blank 80:20 Methanol:Water.    -   4. Cover plate and vortex samples. Centrifuge for 10 minutes at        3300 rpm.    -   5. Transfer 80 μL of supernatant into a clean 96-well plate.    -   6. Add 80 μL of LC-MS water to all wells.    -   7. Cover and vortex.        The concentration of NAD+ in the tissues was determined by        LC/MS. The conditions are as follows:        LC Conditions:        LC: Shimadzu UPLC        Autosampler: Shimadzu SIL-30AC        Analytical Column: Chromolith C18 RP-e 3.0×100 mm        Flow Rate: Variable flow 0.8 mL/min and 1.4 mL/min        Mobile Phases: A: dH₂O B: 1.0% Formic Acid in 50:50 MeOH:ACN        Needle Rinse: dH₂O        Injection Volume: 2.0 μL        LC Gradient Program: A 1.0 min gradient was utilitized going        from 0% to 98% of Mobile        Phase B with a total run time of 3.00 minutes.        Mass Spectrometer Conditions:        Instrument: AB Sciex QTRAP 6500        Scan: Multiple Reaction Monitoring (MRM)        Resolution: Q1 Unit/Q3 Unit        Scan Parameters:

Analyte MRM Transition (m/z) Ionization Mode Alprazolam (IS)309.100/281.100 Da ESI+ NAD 664.000/427.900 Da ESI+

The ratio of mean NAD+ concentration in the liver and skeletal tissuesof treated mice compared to vehicle control mice are given in Table 5.The data is given for the 4, 8, and 24 hour time points.

TABLE 5 4 hr 8 hr 24 hr 4 hr 8 hr 24 hr Compound liver liver liverskeletal skeletal skeletal 1 (Assay 1) 2.6 2.7 0.6 1.2 1.1 1.1 1 (Assay2) 1.6 1.6 0.9 1.4 0.6 0.5 1 (Assay 3) 1.4 1.1 1.3 1.8 1.6 2.3 2 2.0 1.61.8 0.9 1.0 1.4 3 (Assay 1) 1.6 1.6 0.8 0.6 1.4 1.6 3 (Assay 2) 2.1 0.80.9 0.5 0.5 0.4 3 (Assay 3) 1.6 1.5 1.4 1.3 1.0 0.4 4 1.0 1.7 1.8 0.91.4 1.1 6 4.3 6.6 3.5 1.3 1.7 2.6 7 (Assay 1) 1.5 1.7 1.2 1.4 1.1 0.5 7(Assay 2) 1.1 1.1 1.1 1.3 1.2 1.4 8 1.2 1.0 0.9 1.0 0.8 0.8 9 0.8 0.81.3 1.88 1.34 0.96 19 1 1 1 0.8 1.2 0.1 21 2.2 2.1 1.7 1.3 2.4 3.4

The invention claimed is:
 1. A method of treating a disease or disorderassociated with NAD+ biosynthesis, comprising administering a compound,or a stereoisomer or salt thereof, to a subject in need thereof; whereinthe compound has a structure represented by formula I:

wherein V is selected from hydrogen, phenyl and monocyclic heteroaryl,wherein (i) each said monocyclic heteroaryl contains five or six ringatoms of which 1 or 2 ring atoms are heteroatoms selected from N, S, andO, and the remainder of the ring atoms are carbon, and (ii) each saidphenyl or monocyclic heteroaryl is unsubstituted or is substituted byone or two groups independently selected from halogen, trifluoromethyl,C₁-C₆ alkyl, C₁-C₆ alkoxy, and cyano; and wherein the disease ordisorder is a mitochondrial disease or disorder.
 2. The method of claim1, wherein the compound js represented by formula I:

wherein V is independently selected from the following substituents:


3. The method of claim 1, wherein the compound, or a stereoisomer orsalt thereof, is selected from


4. A method of treating a disease or disorder associated with NAD+biosynthesis, comprising administering a compound, or a stereoisomer orsalt thereof, to a subject in need thereof; wherein the compound has astructure represented by formula II:

wherein (a) each R¹ is independently hydrogen; n-alkyl; branched alkyl;cycloalkyl; or aryl, where aryl is optionally substituted with at leastone of C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ alkoxy, F, Cl, Br,I, nitro, cyano, C₁₋₆ haloalkyl, —N(R¹)₂, C₁₋₆ acylamino, —NHSO₂C₁₋₆alkyl, —SO₂N(R^(1′))₂, COR^(1″), and —SO₂C₁₋₆ alkyl; each R^(1′) isindependently hydrogen or C₁₋₂₀alkyl, and R^(1″) is —OR^(1′) or—N(R^(1′))₂; (b) R² is hydrogen, C₁₋₁₀ alkyl or C(O)CR^(3a)R^(3b)NHR¹;or R² and either R^(3a) or R^(3b) together are (CH₂)_(n), forming a ringthat includes the intervening N and C atoms, where n is from 2 to 4; (c)each R^(3a) and R^(3b) are (i) independently selected from hydrogen,C₁₋₁₀ alkyl, cycloalkyl, —(CH₂)c(NR^(3′))₂, C₁₋₆ hydroxyalkyl, —CH₂SH,—(CH₂)₂S(O)_(d)Me, —(CH₂)₃NHC(═NH)NH₂, (1H-indol-3-yl)methyl,(1H-imidazol-4-yl)methyl, —(CH₂)_(e)COR^(3″), aryl and aryl C₁₋₃ alkyl,said aryl groups are optionally substituted with a group selected fromhydroxyl, C₁₋₁₀ alkyl, C₁₋₆ alkoxy, halogen, nitro and cyano; or (ii)R^(3a) and R^(3b) both are C₁₋₆ alkyl; or (iii) R^(3a) and R^(3b)together are (CH₂)_(f) so as to form a spiro ring; or (iv) R^(3a) ishydrogen and R^(3b) and R² together are (CH₂)_(n) forming a ring thatincludes the adjoining N and C atoms; or (v) R^(3b) is hydrogen andR^(3a) and R² together are (CH₂)_(n) forming a ring that includes theadjoining N and C atoms; or (vi) R^(3a) is H and R^(3b) is H, CH₃,CH₂CH₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, CH₂Ph, CH₂-indol-3-yl,CH₂CH₂SCH₃, CH₂CO₂H, CH₂C(O)NH₂, CH₂CH₂COOH, CH₂CH₂C(O)NH₂,CH₂CH₂CH₂CH₂NH₂, CH₂CH₂CH₂NHC(NH)NH₂, CH₂-imidazol-4-yl, CH₂OH,CH(OH)CH₃, CH₂((4′-OH)-Ph), CH₂SH, or C₃₋₇ cycloalkyl; or (vii) R^(3a)is CH₃, CH₂CH₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, CH₂Ph,CH₂-indol-3-yl, —CH₂CH₂SCH₃, CH₂CO₂H, CH₂C(O)NH₂, CH₂CH₂COOH,CH₂CH₂C(O)NH₂, CH₂CH₂CH₂CH₂NH₂, CH₂CH₂CH₂NHC(NH)NH₂, CH₂-imidazol-4-yl,CH₂OH, CH(OH)CH₃, CH₂((4′-OH)-Ph), CH₂SH, or C₃₋₇ cycloalkyl and R^(3b)is H; c is from 1 to 6, d is from 0 to 2, e is from 0 to 3, f is from 2to 5, n is from 2 to 4, and (d) R⁴ is hydrogen; C₁₋₁₀ alkyl optionallysubstituted with alkoxy, di(C₁₋₆ alkyl)-amino, or halogen; C₁₋₁₀haloalkyl; C₃₋₁₀ cycloalkyl; cycloalkyl alkyl; cycloheteroalkyl;aminoacyl; aryl; heteroaryl; substituted aryl; or substitutedheteroaryl; and wherein the disease or disorder is a mitochondrialdisease or disorder.
 5. The method of treatment of claim 4, wherein thecompound, or a stereoisomer or salt thereof, is selected from


6. A method of treating a disease or disorder associated with NAD+biosynthesis, comprising administering a compound, or a stereoisomer orsalt thereof, to a subject in need thereof; wherein the compound has astructure represented by formula III:

wherein each W¹ and W² is independently

(i) each R^(c) and R^(d) is independently H, (C₁-C₈)alkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₃-C₈)carbocyclyl,(C₄-C₈)carbocyclylalkyl, aryl(C₁-C₈)alkyl, heterocyclyl(C₁-C₈)alkyl,(C₆-C₂₀)aryl, (C₂-C₂₀)heterocyclyl or heteroaryl; or (ii) each R^(c) isH and each R^(d) is independently (C₁-C₈)alkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, (C₃-C₈)carbocyclyl, (C₄-C₈)carbocyclylalkyl,aryl(C₁-C₈)alkyl, heterocyclyl(C₁-C₈)alkyl, (C₆-C₂₀)aryl,(C₂-C₂₀)heterocyclyl or heteroaryl; or (iii) each R^(c) is H and eachR^(d) is independently (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₃-C₈)carbocyclyl, (C₄-C₈)carbocyclylalkyl, aryl(C₁-C₈)alkyl,heterocyclyl(C₁-C₈)alkyl, (C₆-C₂₀)aryl, (C₂-C₂₀)heterocyclyl orheteroaryl wherein the chirality of the carbon to which said R^(c) andR^(d) is attached is S; or (iv) each R^(c) is H and each R^(d) isindependently (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₃-C₈)carbocyclyl, (C₄-C₈)carbocyclylalkyl, aryl(C₁-C₈)alkyl,heterocyclyl(C₁-C₈)alkyl, (C₆-C₂₀)aryl, (C₂-C₂₀)heterocyclyl orheteroaryl wherein the chirality of the carbon to which said R^(c) andR^(d) is attached is R; or (v) each R^(c) is H and each R^(d) isindependently (C₁-C₈)alkyl; or (vi) each R^(c) is H and each R^(d) isindependently (C₁-C₈)alkyl wherein the chirality of the carbon to whichsaid R^(c) and R^(d) is attached is S; or (vii) each R^(c) is H and eachR^(d) is independently (C₁-C₈)alkyl wherein the chirality of the carbonto which said R^(c) and R^(d) is attached is R; and each R⁶ isindependently (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₃-C₈)carbocyclyl or (C₄-C₈)carbocyclylalkyl; and wherein the diseaseor disorder is a mitochondrial disease or disorder.
 7. A method oftreating a disease or disorder associated with NAD+ biosynthesis,comprising administering a compound, or a stereoisomer or salt thereof,to a subject in need thereof; wherein the compound is represented byformula III:

wherein W¹ and W² are independently selected from the substituents inTable 1, wherein each R²¹ is independently H or (C₁-C₈)alkyl; each R²²is independently H, R²¹, R²³ or R²⁴, wherein each R²⁴ is independentlysubstituted with 0 to 3 R²³; each R²³ is independently R^(23a), R^(23b),R^(23c) or R^(23d), provided that when R²³ is bound to a heteroatom,then R²³ is R^(23d) or R^(23d); each R^(23a) is independently F, Cl, Br,I, —CN, —N₃ or —NO₂; each R^(23b) is independently Y²¹; each R^(23c) isindependently —R^(2x), —OR^(2x), —N(R^(2x))(R^(2x)), —SR^(2x),—S(O)R^(2x); —S(O)₂R^(2x), —S(O)(OR^(2x)), —S(O)₂(OR^(2x)),—OC(═Y²¹)R^(2x), —OC(═Y²¹)OR^(2x), —OC(═Y²¹)(N(R^(2x))(R^(2x)));—SC(═Y²¹)R^(2x); —SC(═Y²¹)OR^(2x), —SC(═Y²¹)(N(R^(2x))(R^(2x))),—N(R^(2x))C(═Y²¹)R^(2x), —N(R^(2x))C(═Y²¹)OR^(2x), or—N(R^(2x))C(═Y²¹)(N(R^(2x))(R^(2x))); each R^(23d) is independently—C(═Y²¹)R^(2x); —C(═Y²¹)OR^(2x) or —C(═Y²¹)(N(R^(2x))(R^(2x))); eachR^(2x) is independently H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,aryl, heteroaryl; or two R^(2x) taken together with a nitrogen or oxygento which they are both attached form a 3 to 7 membered heterocyclic ringwherein any one carbon atom of said heterocyclic ring can optionally bereplaced with —O—, —S— or —NR²¹—; and wherein one or more of thenon-terminal carbon atoms of each said (C₁-C₈)alkyl may be optionallyreplaced with —O—, —S— or —NR²¹—; each R²⁴ is independently(C₁-C₈)alkyl, (C₂-C₈)alkenyl, or (C₂-C₈)alkynyl; each R²⁵ isindependently R²⁴, wherein each R²⁴ is substituted with 0 to 3 R²³groups; each R^(25a) is independently (C₁-C₈)alkylene,(C₂-C₈)alkenylene, or (C₂-C₈)alkynylene, any one of which said(C₁-C₈)alkylene, (C₂-C₈)alkenylene, or (C₂-C₈)alkynylene is substitutedwith 0-3 R²³ groups; each W²³ is independently W²⁴ or W²⁵; each W²⁴ isindependently R²⁵, —C(═Y²¹)R²⁵, —C(═Y²¹)W²⁵, —SO₂R²⁵, or —SO₂W²⁵; eachW²⁵ is independently carbocycle or heterocycle wherein W²⁵ isindependently substituted with 0 to 3 R²² groups; and each Y²¹ isindependently O or S; and wherein the disease or disorder is amitochondrial disease or disorder.
 8. The method of claim 1, whereintreatment of the mitochondrial disease or disorder comprises increasingmitochondrial metabolism or increasing the activity level of SIRT3. 9.The method of claim 8, wherein increasing the activity level of SIRT3has one or more effects selected from mimicking calorie restriction orexercise, increasing mitochodrial biogenesis, sensitizing a cell toglucose uptake, increasing fatty acid oxidation, decreasing reactiveoxygen species, and promoting cell survival during genotoxic stress. 10.The method of claim 9, wherein increasing the activity level of SIRT3increases metabolic activation of muscles selected from smooth muscles,gut and digestive muscles, skeletal muscles, and cardiac muscles; and/ortreats cachexia and muscle wasting.
 11. The method of claim 8, whereinthe mitochondrial disease is selected from chronic progressive externalophthalmoplegia, myoclonus epilepsy associated with ragged-red fibers,Fukuhara syndrome, Leber's disease, Leigh encephalopathia and Pearson'sdisease.
 12. The method of claim 4, wherein R¹ is optionally substitutedphenyl or naphthyl.
 13. The method of claim 4, wherein R^(1′) is C₁₋₁₀alkyl.
 14. The method of claim 6, wherein

represents an ester of a naturally occurring α-amino acid.
 15. Themethod of claim 6, wherein each R^(c) is H and each R^(d) isindependently (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₃-C₈)carbocyclyl, (C₄-C₈)carbocyclylalkyl, aryl(C₁-C₈)alkyl,heterocyclyl(C₁-C₈)alkyl, (C₆-C₂₀)aryl, (C₂-C₂₀)heterocyclyl orheteroaryl.
 16. The method of claim 6, wherein each R^(c) is H and eachR^(d) is independently (C₁-C₈)alkyl.
 17. The method of claim 6, whereineach R⁶ is independently (C₁-C₈)alkyl.
 18. A method of treating adisease or disorder associated with NAD+ biosynthesis, comprisingadministering a compound, or a stereoisomer or salt thereof, to asubject in need thereof; wherein the compound is represented by formulaIII:

wherein one of W¹ and W² is OR⁵, wherein R⁵ is (C₁-C₈)alkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₃-C₈)carbocyclyl,(C₄-C₈)carbocyclylalkyl, aryl(C₁-C₈)alkyl, heterocyclyl(C₁-C₈)alkyl,(C₆-C₂₀)aryl, (C₂-C₂₀)heterocyclyl or heteroaryl; and the other of W¹and W² is

(i) each R^(c) and R^(d) is independently H, (C₁-C₈)alkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₃-C₈)carbocyclyl,(C₄-C₈)carbocyclylalkyl, aryl(C₁-C₈)alkyl, heterocyclyl(C₁-C₈)alkyl,(C₆-C₂₀)aryl, (C₂-C₂₀)heterocyclyl or heteroaryl; or (ii) each R^(c) isH and each R^(d) is independently (C₁-C₈)alkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, (C₃-C₈)carbocyclyl, (C₄-C₈)carbocyclylalkyl,aryl(C₁-C₈)alkyl, heterocyclyl(C₁-C₈)alkyl, (C₆-C₂₀)aryl,(C₂-C₂₀)heterocyclyl or heteroaryl; or (iii) each R^(c) is H and eachR^(d) is independently (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₃-C₈)carbocyclyl, (C₄-C₈)carbocyclylalkyl, aryl(C₁-C₈)alkyl,heterocyclyl(C₁-C₈)alkyl, (C₆-C₂₀)aryl, (C₂-C₂₀)heterocyclyl orheteroaryl wherein the chirality of the carbon to which said R^(c) andR^(d) is attached is S; or (iv) each R^(c) is H and each R^(d) isindependently (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₃-C₈)carbocyclyl, (C₄-C₈)carbocyclylalkyl, aryl(C₁-C₈)alkyl,heterocyclyl(C₁-C₈)alkyl, (C₆-C₂₀)aryl, (C₂-C₂₀)heterocyclyl orheteroaryl wherein the chirality of the carbon to which said R^(c) andR^(d) is attached is R; or (v) each R^(c) is H and each R^(d) isindependently (C₁-C₈)alkyl; or (vi) each R^(c) is H and each R^(d) isindependently (C₁-C₈)alkyl wherein the chirality of the carbon to whichsaid R^(c) and R^(d) is attached is S; or (vii) each R^(c) is H and eachR^(d) is independently (C₁-C₈)alkyl wherein the chirality of the carbonto which said R^(c) and R^(d) is attached is R; and each R⁶ isindependently (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₃-C₈)carbocyclyl or (C₄-C₈)carbocyclylalkyl; and wherein the diseaseor disorder is a mitochondrial disease or disorder.
 19. The method ofclaim 18, wherein R⁵ is (C₆-C₂₀)aryl, (C₂-C₂₀)heterocyclyl orheteroaryl.
 20. The method of claim 18, wherein R⁵ is unsubstitutedphenyl.
 21. The method of claim 18, wherein R⁵ is pyridyl.
 22. Themethod of claim 18, wherein one of R^(c) or R^(d) is H and the other ofR^(c) or R^(d) is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₃-C₈)carbocyclyl, (C₄-C₈)carbocyclylalkyl, aryl(C₁-C₈)alkyl,heterocyclyl(C₁-C₈)alkyl, (C₆-C₂₀)aryl, (C₂-C₂₀)heterocyclyl orheteroaryl.
 23. The method of claim 18, wherein one of R^(c) or R^(d) isH and the other of R^(c) or R^(d) is (C₁-C₈)alkyl.
 24. The method ofclaim 18, wherein R⁵ is unsubstituted phenyl, one of R^(c) or R^(d) is Hand the other of R^(c) or R^(d) is methyl.
 25. The method of claim 18,wherein R⁶ is (C₁-C₈)alkyl.
 26. The method of claim 18, wherein R⁵ isunsubstituted phenyl and R⁶ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, (C₃-C₈)carbocyclyl, or (C₄-C₈)carbocyclylalkyl.
 27. Themethod of claim 7, wherein the compound, or a stereoisomer or saltthereof, is selected from


28. The method of claim 1, wherein the salt is formed with an anionselected from acetate, triflate, halide, trifluoroacetate, and formate.29. The method of claim 4, wherein the salt is formed with an anionselected from acetate, triflate, halide, trifluoroacetate, and formate.30. The method of claim 6, wherein the salt is formed with an anionselected from acetate, triflate, halide, trifluoroacetate, and formate.31. The method of claim 7, wherein the salt is formed with an anionselected from acetate, triflate, halide, trifluoroacetate, and formate.32. The method of claim 18, wherein the salt is formed with an anionselected from acetate, triflate, halide, trifluoroacetate, and formate.33. The method of claim 1, wherein the salt is formed with an anionselected from OH⁻, H₂PO₄ ⁻, HPO₄ ²⁻, HSO₄ ⁻, SO₄ ²⁻, NO₃ ⁻, HCO₃ ⁻, andCO₃ ²⁻.
 34. The method of claim 4, wherein the salt is formed with ananion selected from OH⁻, H₂PO₄ ⁻, HPO₄ ²⁻, HSO₄ ⁻, SO₄ ²⁻, NO₃ ⁻, HCO₃⁻, and CO₃ ²⁻.
 35. The method of claim 6, wherein the salt is formedwith an anion selected from OH⁻, H₂PO₄ ⁻, HPO₄ ²⁻, HSO₄ ⁻, SO₄ ²⁻, NO₃⁻, HCO₃ ⁻, and CO₃ ²⁻.
 36. The method of claim 7, wherein the salt isformed with an anion selected from OH⁻, H₂PO₄ ⁻, HPO₄ ²⁻, HSO₄ ⁻, SO₄²⁻, NO₃ ⁻, HCO₃ ⁻, and CO₃ ²⁻.
 37. The method of claim 18, wherein thesalt is formed with an anion selected from OH⁻, H₂PO₄ ⁻, HPO₄ ²⁻, HSO₄⁻, SO₄ ²⁻, NO₃ ⁻, HCO₃ ⁻, and CO₃ ²⁻.